Immunology Treatment for Biofilms

ABSTRACT

The invention provides a composition for use in raising an immune response to  P. gingivalis  in a subject, the composition comprising an amount effective to raise an immune response of at least one polypeptide having an amino acid sequence substantially identical to at least 50 amino acids, or an antigenic or immunogenic portion, of one of the polypeptides corresponding to accession numbers selected from the group consisting of AAQ65462, AAQ65742, AAQ66991, AAQ65561, AAQ66831, AAQ66797, AAQ66469, AAQ66587, AAQ66654, AAQ66977, AAQ65797, AAQ65867, AAQ65868, AAQ65416, AAQ65449, AAQ66051, AAQ66377, AAQ66444, AAQ66538, AAQ67117 and AAQ67118. The invention also provides a method of preventing or treating a subject for  P. gingivalis  infection comprising administering to the subject a composition of the invention

FIELD OF THE INVENTION

The present invention relates to compositions and methods for preventingor altering bacterial biofilm formation and/or development such as thosecontaining Porphyromonas gingivalis. In particular the present inventionrelates to the use and inhibition of polypeptides which are regulatedduring growth as a biofilm or under haem-limitation, to modulate biofilmformation and/or development. The present invention relates to theidentification of polypeptides which may be used as the basis for anantibacterial vaccine or an immunotherapeutic/immunoprophylactic.

BACKGROUND OF THE INVENTION

Many bacterial treatments are directed to bacteria in a planktonicstate. However, bacterial pathologies include bacteria in a biofilmstate. For example, Porphyromonas gingivalis is considered to be themajor causative agent of chronic periodontal disease. Tissue damageassociated with the disease is caused by a dysregulated host immuneresponse to P. gingivalis growing as a part of a polymicrobial bacterialbiofilm on the surface of the tooth. Bacterial biofilms are ubiquitousin nature and are defined as matrix-enclosed bacterial populationsadherent to each other and/or to surfaces or interfaces (1). Thesesessile bacterial cells adhering to and growing on a surface as a maturebiofilm are able to survive in hostile environments which can includethe presence of antimicrobial agents, shear forces and nutrientdeprivation.

The Centers for Disease Control and Prevention estimate that 65% ofhuman bacterial infections involve biofilms. Biofilms often complicatetreatment of chronic infections by protecting bacteria from the immunesystem, decreasing antibiotic efficacy and dispersing planktonic cellsto distant sites that can aid reinfection (2,3). Dental plaque is aclassic example of a bacterial biofilm where a high diversity of speciesform a heterogeneous polymicrobial biofilm growing on the surface of thetooth. The surface of the tooth is a unique microbial habitat as it isthe only hard, permanent, non-shedding surface in the human body. Thisallows the accretion of a substantial bacterial biofilm over a lengthytime period as opposed to mucosal surfaces where epithelial cellshedding limits development of the biofilm. Therefore, the changes tothe P. gingivalis proteome that occur between the planktonic and biofilmstates are important to our understanding of the progression of chronicperiodontal disease.

P. gingivalis has been classified into two broad strain groups withstrains including W50 and W83 being described as invasive in animalmodels whilst strains including 381 and ATCC 33277 are described asnon-invasive (4,5). Griffen et al. (6) found that W83/W50-like strainswere more associated with human periodontal disease than other P.gingivalis strains, including 381-like strains, whilst Cutler et al. (7)demonstrated that invasive strains of P. gingivalis were more resistantto phagocytosis than non-invasive strains. Comparison of the sequencedP. gingivalis W83 strain to the type strain ATCC 33277 indicated that 7%of genes were absent or highly divergent in strain 33277 indicating thatthere are considerable differences between the strains (8).Interestingly P. gingivalis strain W50 forms biofilms only poorly undermost circumstances compared to strain 33277 which readily forms biofilms(9). As a consequence of this relatively few studies have been conductedon biofilm formation by P. gingivalis W50.

Quantitative proteomic studies have been employed to determine proteomechanges of human bacterial pathogens such as Pseudomonas aeruginosa,Escherichia coli and Streptococcus mutans from the planktonic to biofilmstate using 2D gel electrophoresis approaches, where protein ratios arecalculated on the basis of gel staining intensity (10-12). Analternative is to use stable isotope labelling techniques such as ICAT,iTRAQ or heavy water (H₂ ¹⁸O) with MS quantification (13). The basis forH₂ ¹⁸O labelling is that during protein hydrolysis endopeptidases suchas trypsin have been demonstrated to incorporate two ¹⁸O atoms into theC-termini of the resulting peptides (14,15). In addition to use in thedetermination of relative protein abundances (16-19), ¹⁸O labelling inproteomics has also been used for the identification of the proteinC-terminus, identification of N-linked glycosylation after enzymaticremoval of the glycan, simplification of MS/MS data interpretation andmore recently for validation of phosphorylation sites (20-23). The¹⁶O/¹⁸O proteolytic labelling method for measuring relative proteinabundance involves digesting one sample in H₂ ¹⁶O and the other samplein H₂ ¹⁸O. The digests are then combined prior to analysis by LC MS/MS.Peptides eluting from the LC column can be quantified by measuring therelative signal intensities of the peptide ion pairs in the MS mode. Theincorporation of two ¹⁸O atoms into the C-terminus of digested peptidesby trypsin results in a mass shift of +4 m/z allowing the identificationof the isotope pairs.

Due to the complexity of the proteome, prefractionation steps areadvantageous for increasing the number of peptide and proteinidentifications. Most prefractionation steps involve a 2D LC approach atthe peptide level after in-solution digestion (24,25). However due topotential sample loss during the initial dehydration steps of theprotein solution, SDS PAGE prefractionation at the protein levelfollowed by ¹⁶O/¹⁸O labelling during in gel digestion has also beencarried out successfully, (26-29). The ¹⁶O/¹⁸O proteolytic labelling isa highly specific and versatile methodology but few validation studieson a large scale have been performed (30). An excellent validation studywas carried out by Qian et al (18) who labelled two similar aliquots ofserum proteins in a 1:1 ratio and obtained an average ratio of 1.02±0.23from 891 peptides. A more recent study by Lane et al (26) furtherdemonstrated the feasibility of the ¹⁶O/¹⁸O method using a reverselabelling strategy to determine the relative abundance of 17 cytochromeP450 proteins between control and cytochrome P450 inducers treated micethat are grafted with human tumours.

SUMMARY OF THE INVENTION

This invention is illustrated by reference to a sample system whereby P.gingivalis W50 is grown in continuous culture and a mature biofilmdeveloped on the vertical surfaces in the chemostat vessel over anextended period of time. The final biofilm is similar to that whichwould be seen under conditions of disease progression, thus allowing adirect comparison between biofilm and planktonic cells. ¹⁶O/¹⁸Oproteolytic labelling using a reverse labelling strategy was carried outafter SDS-PAGE prefractionation of the P. gingivalis cell envelopefraction followed by coupling to off-line LC MALDI TOF-MS/MS foridentification and quantification. Of the 116 proteins identified, 81were consistently found in two independent continuous culture studies.47 proteins with a variety of functions were found to consistentlyincrease or decrease in abundance in the biofilm cells providingpotential targets for biofilm control strategies. Of these 47 proteinsthe present inventors have selected 24 proteins which they believe areparticular useful as targets in treatment and/or prevention of P.gingivalis infection.

Accordingly, the present invention is directed in a first aspect towardsa polypeptide which modulates biofilm formation. In one form, themicroorganisms in the biofilm are bacteria. In one form, the bacteriaisfrom the genus Porphyromonas. In one embodiment, the bacteria is P.gingivalis and the polypeptide has an amino acid sequence selected fromthe group consisting of the sequences corresponding to the accessionnumbers listed in Table 4. The invention extends to sequences at least80% identical thereto, preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical thereto.

The invention also includes a polypeptide corresponding to accessionnumber AAQ65742 (version 0.1) and a polypeptide at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical hereto.

Preferably, the polypeptide is at least 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of any one of the sequencescorresponding to the accession numbers listed in Table 4.

One aspect of the invention is a composition for use in raising animmune response directed against P. gingivalis in a subject, thecomposition comprising an effective amount of at least one polypeptideof the first aspect of the invention or an antigenic or immunogenicportion thereof. The composition may optionally include an adjuvant anda pharmaceutically acceptable carrier. Thus, the composition may containan antigenic portion of such a polypeptide instead of the full lengthpolypeptide. Typically, the portion will be substantially identical toat least 10, more usually 20 or 50 amino acids of a polypeptidecorresponding to the sequences listed in Table 4 and generate animmunological response. In a preferred form, the composition is avaccine.

The invention also provides a composition that raises an immune responseto P. gingivalis in a subject, the composition comprising an amounteffective to raise an immune response of at least one antigenic orimmunogenic portion of a polypeptide corresponding to accession numbersselected from the group consisting of AAQ65462, AAQ65742, AAQ66991,AAQ65561, AAQ66831, AAQ66797, AAQ66469, AAQ66587, AAQ66654, AAQ66977,AAQ65797, AAQ65867, AAQ65868, AAQ65416, AAQ65449, AAQ66051, AAQ66377,AAQ66444, AAQ66538, AAQ67117 and AAQ67118.

In another embodiment, there is provided a composition for use inraising an immune response directed against P. gingivalis in a subject,the composition comprising an effective amount of at least onepolypeptide corresponding to an accession number selected from the groupconsisting of AAQ65462, AAQ66991, AAQ65561 and AAQ66831.

In another embodiment, there is provided a composition for use inraising an immune response directed against P. gingivalis in a subject,the composition comprising an effective amount of a polypeptidecorresponding to accession number AAQ65742.

In another embodiment, there is provided a composition for use inraising an immune response to P. gingivalis in a subject, thecomposition comprising amount effective to raise an immune response ofat least one polypeptide having an amino acid sequence substantiallyidentical to at least 50 amino acids of a polypeptide expressed by P.gingivalis and that is predicted by the CELLO program to beextracellular.

In another embodiment, there is provided a composition for use inraising an immune response to P. gingivalis in a subject, thecomposition comprising an amount effective to raise an immune responseof at least one polypeptide having an amino acid sequence selectedsubstantially identical to at least 50 amino acids of a polypeptide thatcauses an immune response in a mouse or a rabbit.

In one embodiment, there is provided an isolated antigenic polypeptidecomprising an amino acid sequence comprising at least 50, 60, 70, 80, 90or 100 amino acids substantially identical to a contiguous amino acidsequence of one of the sequences corresponding to the accession numberslisted in Table 4. The polypeptide may be purified or recombinant.

In another embodiment there is a composition for the treatment ofperiodontal disease comprising as an active ingredient an effectiveamount of at least one polypeptide of the first aspect of the invention.

In another embodiment there is a composition for the treatment of P.gingivalis infection comprising as an active ingredient an effectiveamount of at least one polypeptide of the first aspect of the invention.

Another aspect of the invention is a method of preventing or treating asubject for periodontal disease comprising administering to the subjecta composition according to the present invention as described above.

Another aspect of the invention is a method of preventing or treating asubject for P. gingivalis infection comprising administering to thesubject a composition according to the present invention as describedabove.

In another aspect of the invention there is a use of a polypeptide ofthe invention in the manufacture of a medicament for the treatment of P.gingivalis infection.

In another aspect of the invention there is a use of a polypeptide ofthe invention in the manufacture of a medicament for the treatment ofperiodontal disease.

The invention also extends to an antibody raised against a polypeptideof the first aspect of the present invention. Preferably, the antibodyis specifically directed against one of the polypeptides correspondingto the accession numbers listed in Table 4. The antibody may be raisedusing the composition for raising an immune response described above.

In one embodiment, there is provided an antibody raised against apolypeptide wherein the polypeptide corresponds to an accession numberselected from the group consisting of AAQ65462, AAQ66991, AAQ65561 andAAQ66831.

In one embodiment, there is provided an antibody raised against apolypeptide wherein the polypeptide corresponds to accession numberAAQ65742.

Another aspect of the invention is a composition useful in theprevention or treatment of periodontal disease, the compositioncomprising an antagonist or combination of antagonists of a P.gingivalis polypeptide of the first aspect of the present invention anda pharmaceutically acceptable carrier, wherein the antagonist(s)inhibits P. gingivalis infection. The antagonist(s) may be an antibody.The invention also includes use of an antagonist or combination ofantagonists in the manufacture of a medicament useful for preventing ortreating periodontal disease.

In a further aspect of the present invention there is provided aninterfering RNA molecule, the molecule comprising a double strandedregion of at least 19 base pairs in each strand wherein one of thestrands of the double stranded region is substantially complementary toa region of a polynucleotide encoding a polypeptide which modulatesbiofilm formation as described above. In one embodiment, one of thestrands is complementary to a region of polynucleotide encoding apolypeptide transcript of the sequences listed in Table 4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: ¹⁶O/¹⁸O quantification of specific BSA ratios. Quantification ofknown amounts of BSA was carried out in the same manner as for thebiofilm and planktonic samples reported in the experimental proceduresto validate the methodology. Briefly pre-determined amounts of BSA wereloaded in adjacent lanes of a NuPAGE gel followed by excision of bandsof equal size, normal or reverse proteolytic labelling, nanoHPLC andMALDI TOF-MS/MS. (A) MS spectra of BSA tryptic peptide, RHPEYAVSVLLR atknown ¹⁶O:¹⁸O labelling ratios 1:1 (i), 2:1 (ii), 1:5 (iii) and 10:1(iv) showing the characteristic doublet isotopic envelope for ¹⁶O and¹⁸O labelled peptide (S0, S2 and S4 are the measured intensities of theisotopic peaks) (B) SDS PAGE gel of known BSA ratios used for thequantification procedure.

FIG. 2: Typical forward and reverse MS and MS/MS spectra from P.gingivalis sample. (i,ii) Zoomed portion of mass spectra showing the[M+H]+ parent precursor ion of the normal and reverse labelled peptideGNLQALVGR belonging to PG2082 and showing the typical 4Da massdifference in a 1:1 ratio (iii,iv) mass spectrum showing the [M+H]+parent precursor ion of the normal and reverse labelled peptideYNANNVDLNR belonging to PG0232 and showing the typical 4Da massdifference in a 2:1 ratio (v, vi) MS/MS spectrum of heavy labelled (+218O) YNANNVDLNR and unlabelled YNANNVDLNR peptide characterized by the 4Da shift of all Y ions.

FIG. 3: Correlation of normal/reverse labelled technical replicates. Log10 transformed scatter plot comparison of peptide abundance ratio of thenormal (Bio18, Plank16) and reverse (Plank18, Bio16) labelling for bothbiological replicates. The abundance ratios of the reverse labelledpeptides have been inversed for a direct comparison. (A) Biologicalreplicate 1 (B) Biological replicate 2

FIG. 4: Distribution and correlation of protein abundances of biologicalreplicates. (A) Normalized average fold change for the 81 quantifiableproteins identified in both biological replicates displayed aGaussian-like distribution. The abundance ratio of each protein wasfurther normalized to zero (R−1) and ratios smaller than 1 were invertedand calculated as (1−(1/R)) (18). All 81 quantifiable proteins from eachbiological replicate were sorted by increasing ratios(Biofilm/Planktonic) and divided equally into six groups with equalnumber of proteins (A-F). Groups C and D represents proteins notsignificantly regulated (<3 SD from 1.0). (B) Distribution of proteinsbased on rankings. Insert: ranking table for the determination ofsimilarity between both biological replicates. Proteins were ranked indescending order with 1 having the highest similarity when bothbiological replicates fell within the same group and 6 having the leastsimilarity.

FIG. 5: Breakdown of the 116 proteins identified in this study based onidentification in one or both biological replicates and number of uniquepeptides identified. The proteins identified from both biologicalreplicates (81) are presented in table 2. Legend shows number of uniquepeptides identified per protein.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention provides method for treating a subject includingprophylactic treatment for periodontal disease. Periodontal diseasesrange from simple gum inflammation to serious disease that results inmajor damage to the soft tissue and bone that support the teeth.Periodontal disease includes gingivitis and periodontitis. Anaccumulation of oral bacteria at the gingival margin causes inflammationof the gums that is called ‘gingivitis.’ In gingivitis, the gums becomered, swollen and can bleed easily. When gingivitis is not treated, itcan advance to ‘periodontitis’ (which means ‘inflammation around thetooth.’). In periodontitis, gums pull away from the teeth and form‘pockets’ that are infected. Periodontitis has a specific bacterialaetiology with P. gingivalis regarded as the major aetiological agentThe body's immune system fights the bacteria as the plaque spreads andgrows below the gum line. If not treated, the bones, gums, andconnective tissue that support the teeth are destroyed. The teeth mayeventually become loose and have to be removed.

Using proteomic a strategy the present inventors identified andquantified the changes in abundance of 116 P. gingivalis cell envelopeproteins between the biofilm and planktonic states, with the majority ofproteins identified by multiple peptide hits. The present inventorsdemonstrated enhanced expression of a large group of cell-surfacelocated C-Terminal Domain family proteins including RgpA, HagA, CPG70and PG99. Other proteins that exhibited significant changes in abundanceincluded transport related proteins (HmuY and IhtB), metabolic enzymes(FrdA and FrdB), immunogenic proteins and numerous proteins with as yetunknown functions.

As will be well understood by those skilled in the art alterations maybe made to the amino acid sequences of the polypeptides that have beenidentified as having a change in abundance between biofilm andplanktonic states. These alterations may be deletions, insertions, orsubstitutions of amino acid residues. The altered polypeptides can beeither naturally occurring (that is to say, purified or isolated from anatural source) or synthetic (for example, by site-directed mutagenesison the encoding DNA). It is intended that such altered polypeptideswhich have at least 85%, preferably at least 90%, 95%, 96%, 97%, 98% or99% identity with the sequences set out in the Sequence Listing arewithin the scope of the present invention. Antibodies raised againstthese altered polypeptides will also bind to the polypeptides having oneof the sequences to which the accession numbers listed in Table 4relate.

Whilst the concept of conservative substitution is well understood bythe person skilled in the art, for the sake of clarity conservativesubstitutions are those set out below.

Gly, Ala, Val, Ile, Leu, Met; Asp, Glu, Ser; Asn, Gln; Ser, Thr;Lys, Arg, His; Phe, Tyr, Trp, His; and Pro, Nα-alkalamino acids.

The practice of the invention will employ, unless otherwise indicated,conventional techniques of chemistry, molecular biology, microbiology,recombinant DNA, and immunology well known to those skilled in the art.Such techniques are described and explained throughout the literature insources such as, J. Perbal, A Practical Guide to Molecular Cloning, JohnWiley and Sons (1984), J. Sambrook et Molecular Cloning: A LaboratoryManual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown(editor), Essential Molecular Biology: A Practical Approach, Volumes 1and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNACloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996),and F. M. Ausubel et al. (Editors), Current Protocols in MolecularBiology, Greene Pub. Associates and Wiley-Interscience (1988, includingall updates until present). The disclosure of these texts areincorporated herein by reference.

An ‘isolated polypeptide’ as used herein refers to a polypeptide thathas been separated from other proteins, lipids, and nucleic acids withwhich it naturally occurs or the polypeptide or peptide may besynthetically synthesised. Preferably, the polypeptide is also separatedfrom substances, for example, antibodies or gel matrix, for example,polyacrylamide, which are used to purify it. Preferably, the polypeptideconstitutes at least 10%, 20%, 50%, 70%, and 80% of dry weight of thepurified preparation. Preferably, the preparation contains a sufficientamount of polypeptide to allow for protein sequencing (ie at least 1,10, or 100 mg).

The isolated polypeptides described herein may be purified by standardtechniques, such as column chromatography (using various matrices whichinteract with the protein products, such as ion exchange matrices,hydrophobic matrices and the like), affinity chromatography utilizingantibodies specific for the protein or other ligands which bind to theprotein.

The terms ‘peptides, proteins, and polypeptides’ are usedinterchangeably herein. The polypeptides of the present invention caninclude recombinant polypeptides including fusion polypeptides. Methodsfor the production of a fusion polypeptide are known to those skilled inthe art.

An ‘antigenic polypeptide’ used herein is a moiety, such as apolypeptide, analog or fragment thereof, that is capable of binding to aspecific antibody with sufficiently high affinity to form a detectableantigen-antibody complex. Preferably, the antigenic polypeptidecomprises an immunogenic component that is capable of eliciting ahumoral and/or cellular immune response in a host animal.

In comparing polypeptide sequences, ‘substantially identical’ means 95%or more identical over its length or identical over any 10 contiguousamino acids.

A ‘contiguous amino acid sequence’ as used herein refers to a continuousstretch of amino acids.

A ‘recombinant polypeptide’ is a polypeptide produced by a process thatinvolves the use of recombinant DNA technology.

A reference to ‘preventing’ periodontal disease means inhibitingdevelopment of the disease condition, but not necessarily permanent andcomplete prevention of the disease.

In determining whether or not two amino acid sequences fall within aspecified percentage limit, those skilled in the art will be aware thatit is necessary to conduct a side-by-side comparison or multiplealignments of sequences. In such comparisons or alignments, differenceswill arise in the positioning of non-identical residues, depending uponthe algorithm used to perform the alignment. In the present context,reference to a percentage identity or similarity between two or moreamino acid sequences shall be taken to refer to the number of identicaland similar residues respectively, between said sequences as determinedusing any standard algorithm known to those skilled in the art. Forexample, amino acid sequence identities or similarities may becalculated using the GAP programme and/or aligned using the PILEUPprogramme of the Computer Genetics Group, Inc., University ResearchPark, Madison, Wis., United States of America (Devereaux et al et al.,1984). The GAP programme utilizes the algorithm of Needleman and Wunsch(1970) to maximise the number of identical/similar residues and tominimise the number and length of sequence gaps in the alignment.Alternatively or in addition, wherein more than two amino acid sequencesare being compared, the Clustal W programme of Thompson et al, (1994) isused.

The present invention also provides a vaccine composition for use inraising an immune response directed against P. gingivalis in a subject,the composition comprising an immunogenically effective amount of atleast one polypeptide of the first aspect of the invention and apharmaceutically acceptable carrier.

The vaccine composition of the present invention preferably comprises anantigenic polypeptide that comprises at least one antigen that can beused to confer a protective response against P. gingivalis. The subjecttreated by the method of the invention may be selected from, but is notlimited to, the group consisting of humans, sheep, cattle, horses,bovine, pigs, poultry, dogs and cats. Preferably, the subject is ahuman. An immune response directed against P. gingivalis is achieved ina subject, when there is development in the host of a cellular and/orantibody-mediated response against the specific antigenic polypeptides,whether or not that response is fully protective.

The vaccine composition is preferably administered to a subject toinduce immunity to P. gingivalis and thereby prevent, inhibit or reducethe severity of periodontal disease. The vaccine composition may also beadministered to a subject to treat periodontal disease wherein theperiodontal disease is caused, at least in part, by P. gingivalis. Theterm ‘effective amount’ as used herein means a dose sufficient to elicitan immune response against P. gingivalis. This will vary depending onthe subject and the level of P. gingivalis infection and ultimately willbe decided by the attending scientist, physician or veterinarian.

The composition of the present invention comprises a suitablepharmaceutically-acceptable carrier, such as a diluent and/or adjuvantsuitable for administration to a human or animal subject. Compositionsto raise immune responses preferably comprise a suitable adjuvant fordelivery orally by nasal spray, or by injection to produce a specificimmune response against P. gingivalis. A composition of the presentinvention can also be based upon a recombinant nucleic acid sequenceencoding an antigenic polypeptide of the present invention, wherein thenucleic acid sequence is incorporated into an appropriate vector andexpressed in a suitable transformed host (eg. E. coli, Bacillussubtilis, Saccharomyces cerevisiae, COS cells, CHO cells and HeLa cells)containing the vector. The composition can be produced using recombinantDNA methods as illustrated herein, or can be synthesized chemically fromthe amino acid sequence described in the present invention.Additionally, according to the present invention, the antigenicpolypeptides may be used to generate P. gingivalis antisera useful forpassive immunization against periodontal disease and infections causedby P. gingivalis.

Various adjuvants known to those skilled in the art are commonly used inconjunction with vaccine formulations and formulations for raising animmune response. The adjuvants aid by modulating the immune response andin attaining a more durable and higher level of immunity using smalleramounts of vaccine antigen or fewer doses than if the vaccine antigenwere administered alone. Examples of adjuvants include incompleteFreunds adjuvant (IFA), Adjuvant 65 (containing peanut oil, mannidemonooleate and aluminium monostearate), oil emulsions, Ribi adjuvant,the pluronic polyols, polyamines, Avridine, Quil A, saponin, MPL, QS-21,and mineral gels such as aluminium salts. Other examples include oil inwater emulsions such as SAF-1, SAF-0, MF59, Seppic ISA720, and otherparticulate adjuvants such as ISCOMs and ISCOM matrix. An extensive butexhaustive list of other examples of adjuvants are listed in Cox andCoulter 1992 [In: Wong W K (ed.) Animals parasite control utilisingtechnology. Bocca Raton; CRC press et al., 1992; 49-112]. In addition tothe adjuvant the vaccine may include conventional pharmaceuticallyacceptable carriers, excipients, fillers, buffers or diluents asappropriate. One or more doses of a composition containing adjuvant maybe administered prophylactically to prevent periodontal disease ortherapeutically to treat already present periodontal disease.

In another preferred composition the preparation is combined with amucosal adjuvant and administered via the oral or nasal route. Examplesof mucosal adjuvants are cholera toxin and heat labile E. coli toxin,the non-toxic B sub-units of these toxins, genetic mutants of thesetoxins which have reduced toxicity. Other methods which may be utilisedto deliver the antigenic polypeptides orally or nasally includeincorporation of the polypeptides into particles of biodegradablepolymers (such as acrylates or polyesters) by micro-encapsulation to aiduptake of the microspheres from the gastrointestinal tract or nasalcavity and to protect degradation of the proteins. Liposomes, ISCOMs,hydrogels are examples of other potential methods which may be furtherenhanced by the incorporation of targeting molecules such as LTB, CTB orlectins (mannan, chitin, and chitosan) for delivery of the antigenicpolypeptides to the mucosal immune system. In addition to thecomposition and the mucosal adjuvant or delivery system the compositionmay include conventional pharmaceutically acceptable carriers,excipients, fillers, coatings, dispersion media, antibacterial andantifungal agents, buffers or diluents as appropriate.

Another mode of this embodiment provides for either a live recombinantviral vaccine, recombinant bacterial vaccine, recombinant attenuatedbacterial vaccine, or an inactivated recombinant viral vaccine which isused to protect against infections caused by P. gingivalis. Vacciniavirus is the best known example, in the art, of an infectious virus thatis engineered to express vaccine antigens derived from other organisms.The recombinant live vaccinia virus, which is attenuated or otherwisetreated so that it does not caused disease by itself, is used toimmunise the host. Subsequent replication of the recombinant viruswithin the host provides a continual stimulation of the immune systemwith the vaccine antigens such as the antigenic polypeptides, therebyproviding long lasting immunity. In this context and below, ‘vaccine’ isnot limited to compositions that raise a protective response butincludes compositions raising any immune response.

Other live vaccine vectors include: adenovirus, cytomegalovirus, andpreferably the poxviruses such as vaccinia (Paoletti and Panicali, U.S.Pat. No. 4,603,112) and attenuated salmonella strains (Stocker et al.,U.S. Pat. Nos. 5,210,035; 4,837,151; and 4,735,801; and Curtis et al. etal., 1988, Vaccine 6: 155-160). Live vaccines are particularlyadvantageous because they continually stimulate the immune system whichcan confer substantially long-lasting immunity. When the immune responseis protective against subsequent P. gingivalis infection, the livevaccine itself may be used in a protective vaccine against P.gingivalis. In particular, the live vaccine can be based on a bacteriumthat is a commensal inhabitant of the oral cavity. This bacterium can betransformed with a vector carrying a recombinant inactivated polypeptideand then used to colonise the oral cavity, in particular the oralmucosa. Once colonised the oral mucosa, the expression of therecombinant protein will stimulate the mucosal associated lymphoidtissue to produce neutralising antibodies. For example, using molecularbiological techniques the genes encoding the polypeptides may beinserted into the vaccinia virus genomic DNA at a site which allows forexpression of epitopes but does not negatively affect the growth orreplication of the vaccinia virus vector. The resultant recombinantvirus can be used as the immunogen in a vaccine formulation. The samemethods can be used to construct an inactivated recombinant viralvaccine formulation except the recombinant virus is inactivated, such asby chemical means known in the art, prior to use as an immunogen andwithout substantially affecting the immunogenicity of the expressedimmunogen.

As an alternative to active immunisation, immunisation may be passive,i.e. immunisation comprising administration of purified immunoglobulincontaining an antibody against a polypeptide of the present invention.

The antigenic polypeptides used in the methods and compositions of thepresent invention may be combined with suitable excipients, such asemulsifiers, surfactants, stabilisers, dyes, penetration enhancers,anti-oxidants, water, salt solutions, alcohols, polyethylene glycols,gelatine, lactose, magnesium stearate and silicic acid. The antigenicpolypeptides are preferably formulated as a sterile aqueous solution.The vaccine compositions of the present invention may be used tocomplement existing treatments for periodontal disease.

The invention also provides a method of preventing or treating a subjectfor periodontal disease comprising administering to the subject avaccine composition according to the present invention. Also provided isan antibody raised against a polypeptide of the first aspect of thepresent invention. Preferably, the antibody is specifically directedagainst the polypeptides of the present invention.

In the present specification the term ‘antibody’ is used in the broadestsense and specifically covers monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies),chimeric antibodies, diabodies, triabodies and antibody fragments. Theantibodies of the present invention are preferably able to specificallybind to an antigenic polypeptide as hereinbefore described withoutcross-reacting with antigens of other polypeptides.

The term ‘binds specifically to’ as used herein, is intended to refer tothe binding of an antigen by an immunoglobulin variable region of anantibody with a dissociation constant (Kd) of 1 μM or lower as measuredby surface plasmon resonance analysis using, for example a BIAcore™surface plasmon resonance system and BIAcore™ kinetic evaluationsoftware (eg. version 2.1). The affinity or dissociation constant (Kd)for a specific binding interaction is preferably about 500 nM to about50 pM, more preferably about 500 nM or lower, more preferably about 300nM or lower and preferably at least about 300 nM to about 50 pM, about200 nM to about 50 pM, and more preferably at least about 100 nM toabout 50 pM, about 75 nM to about 50 pM, about 10 nM to about 50 pM.

It has been shown that the antigen-binding function of an antibody canbe performed by fragments of a full length antibody. Examples of bindingfragments of an antibody include (I) a Fab fragment, a monovalentfragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VHdomains of a single arm of an antibody; (v) a dAb fragment whichconsists of a VH domain, or a VL domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded by separate genes, theycan be joined, using recombinant methods, by a synthetic linker thatenables them to be made as a single protein chain in which the VL and VHregions pair to form monovalent molecules (known as single chain Fv(scFv). Other forms of single chain antibodies, such as diabodies ortriabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites.

Various procedures known in the art may also be used for the productionof the monoclonal and polyclonal antibodies as well as variousrecombinant and synthetic antibodies which can bind to the antigenicpolypeptides of the present invention. In addition, those skilled in theart would be familiar with various adjuvants that can be used toincrease the immunological response, depending on the host species, andinclude, but are not limited to, Freud's (complete and incomplete),mineral gels such as aluminium hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,dinitrophenol, and potentially useful human adjuvants such as BacillusCalmette-Guerin (BCG) and Corynebacterium parvum. Antibodies andantibody fragments may be produced in large amounts by standardtechniques (eg in either tissue culture or serum free using a fermenter)and purified using affinity columns such as protein A (eg for murineMabs), Protein G (eg for rat Mabs) or MEP HYPERCEL (eg for IgM and IgGMabs).

Recombinant human or humanized versions of monoclonal antibodies are apreferred embodiment for human therapeutic applications. Humanizedantibodies may be prepared according to procedures in the literature(e.g. Jones et al. 1986, Nature 321: 522-25; Reichman et al. 1988 Nature332: 323-27; et al. 1988, Science 1534-36). The recently described ‘geneconversion metagenesis’ strategy for the production of humanizedmonoclonal antibody may also be employed in the production of humanizedantibodies (Carter et al. 1992 Proc. Natl. Acad. Sci. U.S.A. 89:4285-89). Alternatively, techniques for generating the recombinant phaselibrary of random combinations of heavy and light regions may be used toprepare recombinant antibodies (e.g. Huse et al. 1989 Science 246:1275-81).

As used herein, the term ‘antagonist’ refers to a nucleic acid, peptide,antibody, ligands or other chemical entity which inhibits the biologicalactivity of the polypeptide of interest. A person skilled in the artwould be familiar with techniques of testing and selecting suitableantagonists of a specific protein, such techniques would includingbinding assays.

The antibodies and antagonists of the present invention have a number ofapplications, for example, they can be used as antimicrobialpreservatives, in oral care products (toothpastes and mouth rinses) forthe control of dental plaque and suppression of pathogens associatedwith dental caries and periodontal diseases. The antibodies andantagonists of the present invention may also be used in pharmaceuticalpreparations (eg, topical and systemic anti-infective medicines).

The present invention also provides interfering RNA molecules which aretargeted against the mRNA molecules encoding the polypeptides of thefirst aspect of the present invention. Accordingly, in a seventh aspectof the present invention there is provided an interfering RNA molecule,the molecule comprising a double stranded region of at least 19 basepairs in each strand wherein one of the strands of the double strandedregion is complementary to a region of an mRNA molecule encoding apolypeptide of the first aspect of the present invention.

So called RNA interference or RNAi is known and further informationregarding RNAi is provided in Hannon (2002) Nature 418: 244-251, andMcManus & Sharp (2002) Nature Reviews: Genetics 3(10): 737-747, thedisclosures of which are incorporated herein by reference.

The present invention also contemplates chemical modification(s) ofsiRNAs that enhance siRNA stability and support their use in vivo (seefor example, Shen et al. (2006) Gene Therapy 13: 225-234). Thesemodifications might include inverted abasic moieties at the 5′ and 3′end of the sense strand oligonucleotide, and a single phosphorthioatelinkage between the last two nucleotides at the 3′ end of the antisensestrand.

It is preferred that the double stranded region of the interfering RNAcomprises at least 20, preferably at least 25, and most preferably atleast 30 base pairs in each strand of the double stranded region. Thepresent invention also provides a method of treating a subject forperiodontal disease comprising administering to the subject at least oneof the interfering RNA molecules of the invention.

The compositions of this invention can also be incorporated in lozenges,or in chewing gum or other products, e.g. by stirring into a warm gumbase or coating the outer surface of a gum base, illustrative of whichare jelutong, rubber latex, vinylite resins, etc., desirably withconventional plasticizers or softeners, sugar or other sweeteners orsuch as glucose, sorbitol and the like.

In a further aspect, the present invention provides a kit of partsincluding (a) a composition of polypeptide inhibitory agent and (b) apharmaceutically acceptable carrier. Desirably, the kit further includesinstructions for their use for inhibiting biofilm formation in a patentin need of such treatment.

Compositions intended for oral use may be prepared according to anymethod known in the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavouringagents, colouring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Throughout this specification the word ‘comprise’, or variations such as‘comprises’ or ‘comprising’, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

All publications mentioned in this specification are herein incorporatedby reference. Any discussion of documents, acts, materials, devices,articles or the like which has been included in the presentspecification is solely for the purpose of providing a context for thepresent invention. It is not to be taken as an admission that any or allof these matters form part of the prior art base or were common generalknowledge in the field relevant to the present invention as it existedin Australia or elsewhere before the priority date of each claim of thisapplication.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive. The invention specifically includes all combinations offeatures described in this specification.

In order that the nature of the present invention may be more clearlyunderstood preferred forms thereof will now be described with referenceto the following Examples.

Growth and Harvesting of P. gingivalis for Biofilm v Planktonic Studies

Porphyromonas gingivalis W50 (ATCC 53978) was grown in continuousculture using a model C-30 BioFlo chemostat (New Brunswick Scientific)with a working volume of 400 mL. Both the culture vessel and mediumreservoir were continuously gassed with 10% CO₂ and 90% N₂. The growthtemperature was 37° C. and the brain heart infusion growth medium(Oxoid) was maintained at pH 7.5. Throughout the entire growth, redoxpotential maintained at −300 mV. The dilution rate was 0.1 h⁻¹, giving amean generation time (MGT) of 6.9 h. Sterile cysteine-HCl (0.5 g/L) andhaemin (5 mg/L) were added. The culture reached steady stateapproximately 10 days after inoculation and was maintained for a further30 days until a thick layer of biofilm had developed on the verticalsurfaces of the vessel.

All bacterial cell manipulations were carried out on ice or at 4° C.During harvesting, the planktonic cells were decanted into a cleancontainer and the biofilm washed twice gently with PGA buffer (10.0 mMNaH₂PO₄, 10.0 mM KCl, 2.0 mM, citric acid, 1.25 mM MgCl₂, 20.0 mM CaCl₂,25.0 mM ZnCl₂, 50.0 mM MnCl₂, 5.0 mM CuCl₂, 10.0 mM CoCl₂, 5.0 mM H₃BO₃,0.1 mM Na₂MoO₂, 10 mM cysteine-HCl with the pH adjusted to 7.5 with 5 MNaOH at 37° C.) followed by harvesting of the biofilm into a 50 mLcentrifuge tube.

Planktonic and biofilm cells were then washed 3 times (7000 g) with PGAbuffer and both samples resuspended to a final volume of 30 mL with washbuffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl₂, pH 8.0, proteinaseinhibitor (Sigma)) and lysed by 3 passages through a French PressPressure Cell (SLM, AMINCO) at 138 MPa. The lysed cells were centrifugedat 2000 g for 30 min to remove any unbroken cells. The supernatant wasfurther centrifuged at 100000 g for 1 h to separate the lysed cells intotheir soluble and insoluble (cell envelope) fractions. The cell envelopefraction was further washed 3 times with wash buffer at 100000 g, for 20min each to remove any soluble contaminations. All samples were thenfrozen and stored at −80° C.

Growth and Harvesting of P. gingivalis for Haem-Limitation and ExcessStudies

P. gingivalis W50 was grown in continuous culture using a Bioflo 110fermenter/bioreactor (New Brunswick Scientific) with a 400 mL workingvolume. The growth medium was 37 g/L brain heart infusion medium (Oxoid)supplemented with 5 mg/mL filter sterilized cysteine hydrochloride, 5.0μg/mL haemin (haem-excess) or 0.1 μg/mL haemin (haem-limited). Growthwas initiated by inoculating the culture vessel with a 24 h batchculture (100 mL) of P. gingivalis grown in the same medium(haem-excess). After 24 h of batch culture growth, the medium reservoirpump was turned on and the medium flow adjusted to give a dilution rateof 0.1 h⁻¹ (mean generation time (MGT) of 6.9 h). The temperature of thevessel was maintained at 37° C. and the pH at 7.4±0.1. The culture wascontinuously gassed with 5% CO₂ in 95% N₂. Cells were harvested duringsteady state growth, washed three times with wash buffer (50 mM Tris-HClpH 8.0, 150 mM NaCl, 5 mM MgCl₂) at 5000 g for 30 min and disrupted with3 passes through a French Pressure Cell (SLM, AMINCO) at 138 MPa. Thelysed cells were then centrifuged at 2000 g for 30 min to removeunbroken cells followed by ultracentrifugation at 100000 g, producing asoluble (supernatant) and membrane fraction. All fractions were carriedout on ice.

Preparation and Analysis of ¹⁸O Proteolytic Labelled Biofilm andPlanktonic Cell Envelope Fraction

The cell envelope fraction was first resuspended in 1 mL of ice coldwash buffer containing 2% SDS, then sonication and vortexing werecarried out to aid resuspension of the pellet. The final step inresuspension involved use of a 29-gauge-insulin needle to help break upparticulates. The mixture was then centrifuged at 40000 g to removeinsoluble particles and the protein concentration of the supernatant wasdetermined using the BCA reagent (Pierce) according to themanufacturer's instructions.

The resuspended samples were subjected to precipitation using 5 volumesof ice cold acetone overnight at −20° C. which further helped toinactivate any proteolytic activity. After acetone precipitation, bothsamples were resuspended to a final concentration of 3 mg/mL with 25 mMTris pH 8.0 and 1% SDS assisted by intermittent sonication, vortexingand the use of a 29-gauge-insulin needle. A second BCA protein assay wasthen carried out to standardize the final protein amount.

Gel electrophoresis on a NuPAGE gel was carried out as permanufacturer's protocol using MOPs running buffer (NuPAGE, Invitrogen)except the samples were boiled at 99° C. for 5 min prior to loading ontoa 10-well 10% NuPAGE gel with MOPs as the running buffer. The biofilmand planktonic samples (30 μg each) were loaded in adjacent lanes on thegel. SDS-PAGE was then carried out at 126 V (constant) at 4° C. untilthe dye front was approximately 1 cm from the bottom of the gel. For thebiological replicate, the gel used was a 4-12% NUPAGE gradient gel usingMOPs as the running buffer to give a similar but not exact pattern ofseparation so as to overcome the potential variation of a protein bandbeing separated into two fractions. Staining was carried out overnightin Coomassie brilliant blue G-250 (31) followed by overnight destainingin ultrapure H₂O.

The two gel lanes were divided into 10 gel bands of equal sizes using acustom made stencil and each section cut into approximately 1 mm³ cubes.Destaining was carried out 3 times in a solution of 50 mM NH₄HCO₃/ACN(1:1). After destaining, the gel cubes were dehydrated with 100% ACN,followed by rehydration/reduction with a solution of 10 mMdithiothreitol in ABC buffer (50 mM NH₄HCO₃) at 56° C. for 30 min. Theexcess solution was removed before adding 55 mM iodoacetamide in ABCbuffer for 60 min at room temperature in the dark. After the alkylationreaction, the gel cubes were washed 3 times in ABC buffer, followed bydehydration twice in 100% ACN for 10 min. The gel cubes were furtherdried under centrifugation using a speedvac for 90 min. Digestion wascarried out in 60 μL solution per gel section containing 2 μg ofsequence grade modified trypsin (Promega) and ½ strength ABC buffer madeup in either H₂ ¹⁶O or H₂ ¹⁸O (H₂ ¹⁸O, >97% purity, Marshall Isotopes)for 20 h at 37° C. After digestion, the peptides were twice extractedfrom the gel using a solution of 50% ACN/0.1% TFA in their respectivewater (H₂ ¹⁶O/H₂ ¹⁸O) and 0.1% TFA with the aid of sonication for 5 mineach. The pooled extract was boiled at 99° C. for 5 min to inactivatethe trypsin followed by freeze drying for 48 h.

The freeze-dried peptides were resuspended in a solution of 5% ACN/0.1%TFA in their respective water (H₂ ¹⁶O/H₂ ¹⁸O) just before analysis usingnanoHPLC and MALDI TOF-MS/MS analysis. The peptide solution (20 μL) wasthen loaded onto an Ultimate Nano LC system (LC Packings) using a FAMOSautosampler (LC Packings) in advanced μL pickup mode. The samples werefirst loaded onto a trapping column (300 μm internal diameter×5 mm) at200 μL/min for 5 min. Separation was achieved using a reverse phasecolumn (LC Packings, C18 PepMap100, 75 μm i.d.×15 cm, 3 μm, 100 Å) witha flow rate of 300 mL/min, and eluted in 0.1% formic acid with an ACNgradient of 0-5 min (0%), 5-10 min (0-16%), 10-90 min (16-80%), 90-100min (80-0%).

Eluents were spotted straight onto pre-spotted anchorchip plates (BrukerDaltonics) using the Proteineer Fc robot (Bruker Daltonics) at 30 sintervals. Prior to spotting, each spot position was pre-spotted with0.2 μL of ultrapure H₂O to reduce the concentration of the acetonitrileduring the crystallization process with the matrix. The plate was washedwith 10 mM ammonium phosphate and 0.1% TFA and air-dried beforeautomated analysis using a MALDI-TOF/TOF (Ultraflex with LIFT IIupgrade, Bruker Daltonics). MS analysis of the digest was initiallycarried out in reflectron mode measuring from 800 to 3500 Da using anaccelerating voltage of 25 kV. All MS spectra were produced from 8 setsof 30 laser shots, with each set needing to have a signal to noise,S/N>6, Resolution >3000 to be included. Calibration of the instrumentwas performed externally with [M+H]⁺ ions of the prespotted internalstandards (Angiotensin II, Angiotensin I, Neurotensin, Renin_substrateand ACTH_Clip) for each group of four samples. LIFT mode forMALDI-TOF/TOF was carried out in a fully automated mode using theFlexcontrol and WarpLC software (Bruker Daltonics). In the TOF1 stage,all ions were accelerated to 8 kV and subsequently lifted to 19 kV inthe LIFT cell and all MS/MS spectra were produced from accumulating 550consecutive laser shots.

Selection of parent precursors was carried out using the WarpLC software(ver 1.0) with the LC MALDI SILE (Stable Isotope Labelling Experiment)work flow. Only the most abundant peak of each heavy or light pairseparated by 4 Da was selected, providing its S/N was >50. Compoundsseparated by less than six LC MALDI fractions were considered the sameand therefore selected only once.

Peak lists were generated using Flexanalysis 2.4 Build 11 (BrukerDaltonics) with the Apex peak finder algorithm with S/N>6. The MS scanwas smoothed once with the Savitzky Golay algorithm using a width of 0.2m/z and baseline subtraction was achieved using the Median algorithmwith flatness of 0.8.

Protein identification was achieved using the MASCOT search engine(MASCOT version 2.1.02, Matrix Science) on MS/MS data queried againstthe P. gingivalis database obtained from The Institute for GenomicResearch (TIGR) website (www.tigr.org). MASCOT search parameters were:charge state 1+, trypsin as protease, one missed cleavage allowed and atolerance of 250 ppm for MS and 0.8 m/z for MS/MS peaks. Fixedmodification was set for carbamidomethyl of cysteine and variablemodification was C-terminal ¹⁸O labelled lysine and arginine residues.

A reverse database strategy as described previously (32) was employed todetermine the minimum peptide MASCOT score required to omit falsepositives for single peptide identification. Briefly, the databaseconsists of both the sequence of every predicted P. gingivalis proteinin its normal orientation and the same proteins with their sequencereversed (3880 sequences). The whole MS/MS dataset was then searchedagainst the combined database to determine the lowest Mascot score togive 0% false positives. A false positive was defined as a positivematch to the reversed sequence (bold red and above peptide thresholdscore). A false positive rate for single hit peptides was determined tobe 0.5% with Mascot peptide ion scores of >threshold and <25. When theMascot peptide ion score was >30, there was no match to the reversedatabase. In order to increase the confidence of identification forsingle hits peptide, we used a minimum Mascot peptide ion score of >50which gives a two order of magnitude lower probability of incorrectidentification than if a score of 30 was used, according to the Mascotscoring algorithm.

The matched peptides were evaluated using the following criteria, i) atleast 2 unique peptides with a probability based score corresponding toa p-value <0.05 were regarded as positively identified (required boldred matches) where the score is −log X 10 log(P) and P is theprobability that the observed match is a random event (33), ii) whereonly one unique peptide was used in the identification of a specificprotein (identification of either heavy or light labelled peptide isconsidered as one) the MASCOT peptide ion score must be above 50 or thatpeptide is identified in more than one of the four independentexperiments (2 biological replicates and 2 technical replicates).

Due to the mixed incorporation of one or two ¹⁸O atoms into thepeptides, the contribution of the natural abundance of the ¹⁸O isotopeand the H₂ ¹⁸O purity (a=0.97), the ratios of the peptides R weremathematically corrected using equation:

R=(I ₁ +I ₂)/I₀  (1)

I₀, I₁ and I₂ were calculated according to the following equations (27),

$\begin{matrix}{I_{1} = \frac{{a\; S_{2}} - {\left\lbrack {{aJ}_{2} - {2\left( {1 - a} \right)J_{4}}} \right\rbrack S_{0}} - {2\left( {1 - a} \right)S_{4}}}{a^{2} - {\left( {2 - a - a^{2}} \right)J_{2}} + {2\left( {1 - a} \right)^{2}J_{4}}}} & (2) \\{I_{0} = {S_{0} - {\left( {1 - a} \right)I_{1}}}} & (3) \\{I_{2} = {\frac{1}{a^{2}}\left( {S_{4} - {J_{4}I_{0}} - {J_{2}I_{1}}} \right)}} & (4)\end{matrix}$

Where S₀, S₂ and S₄ are the measured intensities of the monoisotopicpeak for peptide without ¹⁸O label, the peak with 2 Da higher than themonoisotopic peak, and the peak with 4Da higher than the monoisotopicpeak respectively (FIG. 1A). J₀, J₂ and J₄ are the correspondingtheoretical relative intensities of the isotopic envelope of the peptidecalculated from MS-Isotope (http://prospector.ucsf.edu). However whenthe intensity of the second isotopic peaks (S₁ and S₅) was more intensethan the first isotopic peaks (S₀ and S₄), the ratio was simplycalculated as S₁ divided by S₅. This was true especially for largepeptides above 2000 m/z where the contribution of the fifth isotopicpeak of the ¹⁶O labelled peptide to the S₄ peak becomes significant.Calculation of mixed ¹⁶O¹⁸O incorporation was determined by thedifference in the experimental S₂ and theoretical S₂ (J₂) as apercentage of experimental S₄.

Protein abundance ratios were determined by averaging all identifiedpeptides of the same protein, even when the same protein was identifiedin more than one gel section. The data from each ‘normal’ replicate wascombined with the inversed ratios from its respective ‘reverse’replicate providing an average ratio and standard error for each proteinin each biological replicate. Normalization of both the biologicalreplicates was then carried out similarly to that previously reported(34,35). Briefly the averaged ratio for each biological replicate wasmultiplied by a factor so that the geometric mean of the ratios wasequal to one.

Preparation and Analysis of ICAT Labelled Haem-Limited and Excess Cells

Protein labelling and separation were based on the geLC-MS/MS approach(Li et al., 2003) using the cleavable ICAT reagent (Applied Biosystems).Another proteomic approach has been taken in PCT/AU2007/000890 which isherein incorporated by reference. Protein was first precipitated usingTCA (16%) and solubilised with 6 M urea, 5 mM EDTA, 0.05% SDS and 50 mMTris-HCl pH 8.3. Protein concentration was determined using the BCAprotein reagent and adjusted to 1 mg/ml. 100 μg of protein from eachgrowth condition was individually reduced using 2 μL of 50 mMTris(2-carboxy-ethyl)phosphine hydrochloride for 1 h at 37° C. Reducedprotein from the haem-limitation growth condition was then alkylatedwith the ICAT_(heavy) reagent and protein from haem-excess growthcondition with the ICAT_(light) reagent. The two samples were thencombined and subjected to SDS-PAGE on a precast Novex 10% NUPAGE gel(Invitrogen). The gel was stained for 5 min using SimplyBlue™ SafeStain(Invitrogen) followed by destaining with water. The gel lane was thenexcised into 20 sections from the top of the gel to the dye front.

The excised sections were further diced into 1 mm³ cubes and in-geldigested overnight and extracted twice according to the above procedure.The pooled supernatant was dried under reduced vacuum to about 50 μLfollowed by mixing with 500 μL of affinity load buffer before loadingonto the affinity column as per manufacturer's instruction (AppliedBiosystems). Eluted peptides were dried and the biotin tag cleaved withneat TFA at 37° C. for 2 h followed by drying under reduced vacuum. Thedried samples were suspended in 35 μL of 5% acetonitrile in 0.1% TFA.

MS was carried out using an Esquire HCT ion trap mass spectrometer(Bruker Daltonics) coupled to an UltiMate Nano LC system (LCPackings—Dionex). Separation was achieved using a LC Packings reversedphase column (C18 PepMap100, 75 μm i.d.×15 cm, 3 μm, 100 Å), and elutedin 0.1% formic acid with the following acetonitrile gradient: 0-5 min(0%), 5-10 min (0-10%), 10-100 min (10-50%), 100-120 min (50-80%),120-130 min (80-100%).

The LC output was directly interfaced to the nanospray ion source. MSacquisitions were performed under an ion charge control of 100000 in them/z range of 300-1500 with maximum accumulation time of 100 ms. Whenusing GPF three additional m/z ranges (300-800, 700-1200 and 1100-1500)were used to select for precursor ions and each m/z range was carriedout in duplicate to increase the number of peptides identified. MS/MSacquisition was obtained over a mass range from 100-3000 m/z and wasperformed on up to 10 precursors for initial complete proteome analysisand 3 for ICAT analysis for the most intense multiply charged ions withan active exclusion time of 2 min.

Peak lists were generated using DataAnalysis 3.2 (Bruker Daltonics)using the Apex peak finder algorithm with a compound detection thresholdof 10000 and signal to noise threshold of 5. A global charge limitationof +2 and +3 were set for exported data. Protein identification wasachieved using the MASCOT search engine (MASCOT 2.1.02, Matrix Science)on MS/MS data queried against the P. gingivalis database obtained fromThe Institute for Genomic Research (TIGR) website (www.tigr.org). Thematched peptides were further evaluated using the following criteria, i)peptides with a probability based Mowse score corresponding to a p-valueof at most 0.05 were regarded as positively identified, where the scoreis −log X 10(log(P)) and P is the probability that the observed match isa random event ii) where only one peptide was used in the identificationof a specific protein and the MASCOT score was below 30, manualverification of the spectra was performed. To increase confidence in theidentification of ICAT-labelled proteins especially for those withsingle peptide hits, additional filters were applied as follows: i) theheavy and light peptides of an ICAT pair must have exhibited closelyeluting peaks as determined from their extracted ion chromatograms ii)for proteins with a single unique peptide, this peptide must have beenidentified more than once (e.g in different SDS-PAGE fractions or inboth the light and heavy ICAT forms iii) if a single peptide did notmeet the criteria of (ii), the MASCOT score must have been ≧25, theexpectation value ≦0.01 and the MS/MS spectrum must have exhibited acontiguous series of ‘b’ or ‘y’-type ions with the intense ions beingaccounted. Determinations of false positives were as described above.

The ratio of isotopically heavy ¹³C to light ¹²C ICAT labelled peptideswas determined using a script from DataAnalysis (Bruker Daltonics) andverified manually based on measurement of the monoisotopic peakintensity (signal intensity and peak area) in a single MS spectrum. Theminimum ion count of parent ions used for quantification was 2000although >96% of both heavy and light precursor ions were >10000. In thecase of poorly resolved spectra, the ratio was determined from the areaof the reconstructed extracted ion chromatograms (EIC) of the parentions. Averages were calculated for multiple peptides derived from asingle parent protein and outliers were removed using the Grubb's testwith a=0.05.

The cellular localization of P. gingivalis proteins was predicted usingCELLO (http://cello.life.nctu.edu.tw (36)). Extracellular, outermembrane, inner membrane and periplasmic predictions were considered tobe from the envelope fraction.

The concentrations of short-chain fatty acids (SCFA) in cell-freeculture supernatants (uninoculated, haem-excess and haem-limited) weredetermined by capillary gas chromatography based on the derivatizationmethod of Richardson et al. (37).

The correlation coefficient (r) between both biological replicates wasevaluated using the Pearson correlation coefficient function fromMicrosoft Excel. The coefficient of variance (CV) was calculated by thestandard deviation of the peptide abundance ratios divided by the mean,expressed as a percentage.

Extraction of Nucleic Acids for Transcriptomic Analysis

RNA was extracted from 5 mL samples of P. gingivalis cells harvesteddirectly from the chemostat. To each sample 0.2 volumes of RNAStabilisation Reagent (5% v/v phenol in absolute ethanol) were added.Cells were pelleted by centrifugation (9000 g, 5 min, 25° C.),immediately frozen in liquid nitrogen and stored at −70° C. for laterprocessing. Frozen cells were suspended in 1 mL of TRIzol reagent(Invitrogen) per 1×10¹⁰ cells and then disrupted using Lysing Matrix Bglass beads (MP Biomedicals) and the Precellys 24 homogeniser (BertinTechnologies, France). The glass beads were removed by centrifugationand the RNA fraction purified according to the TRIzol manufacturer's(Invitrogen) protocol, except that ethanol (at a final concentration of35%) rather than isopropanol was added at the RNA precipitation stageand samples were then transferred to the spin-columns from the IllustraRNAspin Mini RNA Isolation kit (GE Healthcare). RNA was purifiedaccording to the manufacturer's instructions from the binding steponwards, including on-column DNAse treatment to remove any residual DNA.RNA integrity was determined using the Experion automatedelectrophoresis station (Bio-Rad).

Genomic DNA was extracted from P. gingivalis cells growing in continuousculture using the DNeasy Blood & Tissue Kit (Qiagen) in accordance withthe manufacturer's instructions.

Microarray Design, Hybridization and Analysis

Microarray slides were printed by the Australian Genome ResearchFacility and consisted of 1977 custom designed 60-mer oligonucleotideprobes for the predicted protein coding regions of the P. gingivalis W83genome including additional protein coding regions predicted by the LosAlamos National Laboratory Oralgen project. Microarray Sample Pool (MSP)control probes were included to aid intensity-dependent normalisation.The full complement of probes was printed 3 times per microarray slideonto Corning UltraGAPS coated slides.

Slides were hybridised using either haeme-excess or heme-limited sampleslabelled with Cy3, combined with a universal genomic DNA referencelabelled with Cy5 (GE Lifesciences). cDNA was synthesized from 10 μg oftotal RNA using the SuperScript plus indirect cDNA labelling system(Invitrogen), with 5 μg of random hexamers (Invitrogen) for priming ofthe cDNA synthesis reaction. cDNA was labelled with Cy3 using theAmersham CyDye post-labelling reactive dye pack (GE Lifesciences) andpurified using the purification module of the Invitrogen labellingsystem. Cy5-dUTP labelled genomic cDNA was synthesized in a similarmanner from 400 ng of DNA, using the BioPrime Plus Array CGH IndirectGenomic Labelling System (Invitrogen).

Prior to hybridisation, microarray slides were immersed for 1 h inblocking solution (35% formamide, 1% BSA, 0.1% SDS, 5×SSPE [1×SSPE is150 mM NaCl, 10 mM NaH₂PO₄, 1 mM EDTA]) at 42° C. After blocking slideswere briefly washed in H₂O followed by 99% ethanol and then dried bycentrifugation. Labelled cDNAs were resuspended in 55 μL ofhybridization buffer (35% formamide, 5×SSPE, 0.1% SDS, 0.1 mg mL⁻¹Salmon Sperm DNA) denatured at 95° C. for 5 min then applied to slidesand covered with LifterSlips (Erie Scientific). Hybridisation wasperformed at 42° C. for 16 h. Following hybridisation slides weresuccessively washed in 0.1% SDS plus 2×SSC [1×SSC is 150 mM NaCl 15 mMsodium citrate] (5 min at 42° C., all further washes performed at roomtemperature), 0.1% SDS plus 0.1×SSC (10 min), 0.1×SSC (4 washes, 1 mineach), and then quickly immersing in 0.01×SSC, then 99% ethanol andusing centrifugation to dry the slides.

Slides were scanned using a GenePix 4000B microarray scanner and imagesanalysed using GenePix Pro 6.0 software (Molecular Devices). Threeslides were used for each treatment (haeme-limitation or haeme-excess)representing three biological replicates.

Image analysis was performed using the GenePix Pro 6.0 software(Molecular Devices), and “morph” background values were used as thebackground estimates in further analysis. To identify differentiallyexpressed genes the LIMMA software package was used with a cut off ofP<0.005. Within array normalisation was performed by fitting a globalloess curve through the MSP control spots and applying the curve to allother spots. The Benjamini Hochberg method was used to control the falsediscovery rate to correct for multiple testing.

Gene predictions were based on the P. gingvalis W83 genome annotationfrom the The Institute for Genomic Research (TIGR, www.tigr.orq). Operonprediction was carried out from the Microbesonline website(http://microbesonline.org)

Response of P. gingivalis to Haeme-Limitation as Determined Using DNAMicroarray Analysis

A DNA microarray analysis of the effect of haeme-limited growth on P.gingivalis global gene expression was carried out under identical growthconditions employed for the proteomic analysis. Analysis of data fromthree biological replicates identified a total of 160 genes that showedstatistically significant differential regulation between haeme-excessand haeme-limitation, with the majority of these genes showing increasedlevels of expression under conditions of heme-limitation and only 8genes being down-regulated. Many of the up-regulated genes werepredicted to be in operons and the majority of these showed similarchanges in transcript levels (Table 3 and 5). There was broad agreementbetween the transcriptomic and proteomic data with a significantcorrelation between the two data sets where differential regulation uponhaeme-limitation was observed [Spearman's correlation 0.6364, p<0.05].However for some of the proteins showing differences in abundance fromthe proteomic analysis, the transcriptomic analysis of the correspondinggenes did not detect any statistically significant differences in theabundance of the mRNA. The microarray analyses tended to identify onlythose genes encoding proteins that had large changes in abundance asdetermined by the proteomic analysis (Tables 3 and 5). Where protein andtranscript from the same gene were found to be significantly regulatedby haeme-limitation the majority showed the same direction ofregulation. The exceptions were two gene products, PG0026 a CTD familyputative cell surface proteinase and PG2132 a fimbrillin (FimA). Theseproteins decreased in abundance in the proteomic analysis underhaeme-limitation but were predicted to be up-regulated by thetranscriptomic analysis. Both these proteins are cell surface locatedand it is quite possible that they are either released from the cellsurface or post-translationally modified which could preclude them frombeing identified as up-regulated in the proteomic analysis.

In addition to the gene products discussed in more detail belowtranscription of several genes of interest were significantlyup-regulated including the genes of a putative operon of two genes,PG1874 and PG1875, one of which encodes Haemolysin A; eight concatenatedgenes PG1634-PG1641 of which PG1638 encodes a putative thioredoxin andPG1043 that encodes FeoB2, a manganese transporter. PG1858 which encodesa flavodoxin was the most highly up-regulated gene at 15.29-fold. Of the152 significantly up-regulated genes ˜55 have no predicted function.

Continuous Culture and Biofilm Formation

P. gingivalis W50 was cultured in continuous culture over a 40 dayperiod during which the cell density of the culture remained constantafter the first 10 days with an OD₆₅₀ of 2.69±0.21 and 2.80±0.52 forbiological replicates 1 and 2 respectively. This equates to a celldensity of ˜3 mg cellular dry weight/mL. Over this time period a biofilmof P. gingivalis cells developed on the vertical glass wall of thefermenter vessel. This biofilm was ˜2 mm thick at the time of harvest.

Validation of ¹⁶O/¹⁸O Quantification Method Using BSA

To determine the accuracy and reproducibility of the ¹⁶O/¹⁸Oquantification method, known amounts of BSA were loaded onto adjacentgel lanes to give ratios of 1:1, 1:2, 1:5 and 10:1 (FIG. 1B). The bandswere subjected to in-gel tryptic digestion in the presence of either H₂¹⁶O or H₂ ¹⁸O, mixed and then analyzed by LC MALDI-MS/MS. A typical setof spectra for a single BSA tryptic peptide across the four ratios showsthe preferential incorporation of two ¹⁸O atoms, which is seen mostclearly by the predominance of the +4 Da peak in the 10:1 BSA ratio, andby the almost symmetrical doublet in the 1:1 spectrum, simplifying bothquantification and identification (FIG. 1A). The average incorporationof a single ¹⁸O atom was estimated to be <7% based on the 1:1 labelling(Supplementary Table). The calculated average ratios for all identifiedBSA peptides were 0.98±0.12, 2.22±0.26, 4.90±0.75 and 10.74±2.04 forratios of 1:1 (triplicate), 2:1 (and 1:2), 1:5 and 10:1, respectivelyindicating a good dynamic range, high accuracy of ±2-11% and a low CVranging from 11.75% to 18.95% (Table 1). The reproducible accuracy ofthe 1:1 mixture (performed in triplicate) implies that labelling biaswas very low. This was further confirmed by comparing normal and reverselabelled BSA at a 2:1 ratio, using only peptides that were identified inboth experiments. The normal ratio was determined to be 2.11±0.33 whilethe reverse was 2.30±0.20 (Table 1).

Experimental Design for Quantitative Analysis of Biofilm and PlanktonicSamples

The design of this study involved the use of two biological replicates,that is two independent continuous cultures, each one split into abiofilm sample obtained from the walls of the vessel, and a planktonicsample obtained from the fluid contents of the vessel. Two technicalreplicates for each biological replicate were performed, and although wehad established that there was no significant labelling bias with BSA,we chose to utilize the reverse labelling strategy as there is a lack of¹⁶O/¹⁸O labelling validation studies that have been conducted on complexbiological samples (30). Therefore in total there were four experiments,each consisting of 10 LC-MALDI MS/MS runs stemming from 2×10 gelsegments.

FIG. 2 shows typical MS and MS/MS spectra of two normal and reverselabelled peptides from the biofilm/planktonic samples illustrating thetypical reverse labelling pattern. As with the BSA data, it could beseen that there was a high level of double ¹⁸O incorporation with theaverage mixed incorporation calculated to be <15% for all peptides,confirming that the ¹⁶O/¹⁸O proteolytic labelling method was alsoeffective with complex samples (data not shown). The predominance ofdoubly labelled peptides was further confirmed by the relatively fewMascot hits to the +2 Da species. MS/MS spectra of the heavy labelledpeptides further revealed the expected +4 Da shifts in the Y ions (FIG.2).

The Cell Envelope Proteome of Planktonic and Mature Biofilm P.gingivalis Cells

We have identified and determined the relative abundance of 116 proteinsfrom 1582 peptides based on the selection criteria described in theexperimental procedures section. Of the proteins identified, 73.3% wereidentified by more than 2 unique peptides, 12.9% were from 1 uniquepeptide but identified in both biological replicates and 13.8% wereidentified only by 1 unique peptide with Mascot peptide ion score of >50(FIG. 5). CELLO (36) predicted 77.6% of these proteins to be from thecell envelope thereby showing the effectiveness of this cell envelopeenrichment method. Bioinformatics classification by TIGR (www.tigr.org)and ORALGEN oral pathogen sequence databases (www.oralgen.lanl.gov)predicted a large percentage of the identified proteins to be involvedin transport, have proteolytic activities, or cell metabolism functions.Interestingly 55% of all identified proteins were of unknown functions.

To compare technical replicates of the biological data, the Log₁₀transformed protein abundance ratios of each pair of normal and reverselabelled experiments were plotted against each other (FIG. 3). Linearregression of these plots indicated that each pair is highly correlatedwith R² values of 0.92 and 0.82 for biological replicate 1 and 2,respectively. The slope of each linear fit was also similar to theexpected value of 1.0 at 0.97 and 0.93 for biological replicate 1 and 2,respectively indicating no labelling bias between the technicalreplicates (FIG. 3). The protein abundance ratios from the technicalreplicates were averaged to give a single ratio for each biologicalreplicate.

Before comparing the average data for the two biological replicates, theprotein abundance ratios of each biological replicate were normalized togive an average mean ratio of 1.0. A plot of the normalized proteinabundance ratios from both the biological replicates exhibits aGaussian-like distribution closely centered at zero (FIG. 4A) similar tothat described by others (40,41). There was a significant positivecorrelation between the two biological replicates (Pearson's correlationcoefficient r=0.701, p<0.0001) indicating that the growth of thebiofilm/planktonic cultures and all downstream processing of the samplescould be reproduced to a satisfactory level. To determine which proteinswere consistently regulated in the two biological replicates, a simpleranking chart was constructed where proteins were divided into 6 groups(A-F) according to their abundance ratio and then ranked 1-6 accordingto group-based correlation, with those ranked 1 having the highestsimilarity when a protein from both biological replicates fell withinthe same group (FIG. 4B). Using the ranking chart, we were able todetermine that 34 out of 81 (42%) of the proteins identified from bothreplicates were ranked number one, considerably higher than the valueexpected for a random correlation which would be 17% (or ⅙). Themajority of the remaining proteins were ranked number two, and thereforein total, 70 proteins (86.4%) were considered to be similarly regulatedbetween the two experiments (ranked 1 or 2; Table 2).

Based on the measured standard deviation (±0.26) of the 2:1 BSAlabelling experiment (Table 1), protein abundance changes were deemed tobe biologically significant when they differed from 1.0 by >3 standarddeviations (either >1.78 or <0.56) (18,42). Using this criteria, theabundance of 47 out of the 81 proteins identified in both replicateswere significantly changed (based on the average ratios), and of these,42 were ranked either 1 or 2 (Table 2). Of the 42 proteins ranked 1 and2, 24 had significantly increased in abundance and 18 had decreased inabundance.

Enzymes of Metabolic Pathways Showing Co-Ordinated Regulation

Twenty proteins involved in the glutamate/aspartate catabolism wereidentified in the haem-limited vs haem-excess study using ICAT labellingstrategies (Table 3). Of those, enzymes catalyzing six of the eightsteps directly involved in the catabolism of glutamate to butyrate wereidentified and found to have increased 1.8 to 4 fold underhaem-limitation (Table 3). Although the other two catalytic enzymes(PG0690, 4-hydroxybutyrate CoA-transferase and PG1066,butyrate-acetoacetate CoA-transferase) were not detected using ICAT,they were found to be present in a separate qualitative study atcomparable high ion intensities to those proteins reported in Table 3(not shown) and belong to operons shown to be upregulated. On the otherhand, the effect of haem-limitation on the abundances of the enzymes ofthe aspartate catabolic pathway was mixed, with the enzymes catalyzingthe breakdown of aspartate to oxaloacetate in the oxidative degradationpathway being unchanged and the enzymes involved in the conversion ofpyruvate to acetate showing an increase of 2 to 4.4 fold.

The abundance of two iron containing fumarate reductase enzymes, FrdA(PG1615) and FrdB (PG1614) that together catalyse the conversion offumarate to succinate via the reductive pathway from aspartate, wassignificantly reduced in cells cultured in haem-limitation (Table 3).These two proteins, that are encoded in an operon (Baughn et al., 2003),show similar changes in abundance in response to haem-limitation (FrdAL/E=0.35; FrdB L/E=0.25).

Analysis of Organic Acid End Products

The amounts of acetate, butyrate and propionate in the spent culturemedium of P. gingivalis grown under haem limitation were 13.09±1.82,7.77±0.40 and 0.71±0.05 mmole/g cellular dry weight, respectively.Levels of acetate, butyrate and propionate in the spent culture mediumof P. gingivalis grown in haem excess were 6.00±0.36, 6.51±0.04 and0.66±0.07 mmole/g cellular dry weight, respectively.

The above results illustrate the changes in protein abundance that occurwhen planktonic P. gingivalis cells adhere to a solid surface and growas part of a mature monospecies biofilm. It is the first comparativestudy of bacterial biofilm versus planktonic growth to utilize eitherthe geLC MS approach of Gygi's group (46) or the ¹⁶O/¹⁸O proteolyticlabelling method to determine changes in protein abundances as all othersuch studies published to date have utilized 2D gel electrophoresisbased methods (10-12). A two technical replicate and two biologicalreplicate ¹⁶O/¹⁸O reverse labelling approach was successfully employedto quantitate and validate the changes in protein abundance.

Continuous Culture of P. gingivalis

In this study P. gingivalis W50 was cultured in continuous culture asopposed to the more traditional methodology of batch culture. Batchculture introduces a large range and degree of variation into bacterialanalyses due to interbatch variables such as: size and viability of theinoculum, exact growth stage of the bacterium when harvested, levels ofavailable nutrients in the medium and redox potential of the medium,amongst other factors. In continuous culture the bacterium is grown formany generations under strictly controlled conditions that includegrowth rate, cell density, nutrient concentrations, temperature, pH andredox potential. (44,47,48). A previous study has demonstrated a highlevel of reproducibility of Saccharomyces cerevisiae transcriptomicanalyses continuously cultured in chemostats in different laboratories(49). Furthermore in our study the growth of both biofilm and planktoniccells was carried out in a single fermentation vessel, reducingvariability as compared to separate cultivations. The consistent changesin P. gingivalis cell envelope protein abundances between biologicalreplicates of 86.4% of the identified proteins (ranked 1 and 2) seen inthis study illustrate the applicability of the continuous culture systemand the ¹⁶O/¹⁸O proteolytic labelling strategy to the analysis of theeffect of biofilm growth on the P. gingivalis proteome.

Efficiency of ¹⁸O Labelling

The basic proteomic method employed in this study was the geLC MS method(46,50) due to the high resolution and solubility of membrane proteinsthat the SDS-PAGE method affords. This method was combined with a single¹⁸O labelling reaction during the in-gel digestion procedure similar tothat described by others (26-29). Efficient labelling should result inthe incorporation of two ¹⁸O atoms into the C-terminus of each peptideand should be resistant to back-exchange with ¹⁶O. This was found to bethe case in our study with BSA where the level of single ¹⁸O atomincorporation was estimated to be <7% and the mean ratios obtained forvarious BSA experiments were found not to significantly favour ¹⁶O(Table 1) suggesting that back exchange with normal water was not aproblem. Similar results were also obtained for the biological samples.A crucial step for efficient ¹⁸O labelling was the need for the completeremoval of the natural H₂ ¹⁶O followed by resolubilization of theprotein in H₂ ¹⁸O before tryptic digestion employing a‘single-digestion’ method. Although a number of studies have used a‘double digestion’ method (51,52), the single digestion method has theadvantage of giving a higher efficiency of ¹⁸O labelling as in thedouble digestion method some tryptic peptides were unable to exchangeeither of their C-terminal ¹⁶O atoms for an ¹⁸O atom after the initialdigestion (53). We further utilized an in-gel digestion method where theprotein is retained in the gel matrix during the initial dehydrationstep using organic solvents as in any standard in-gel digestionprotocol. Complete removal of any trace natural H₂ ¹⁶O was achievedthrough lyophilization by centrifugation under vacuum while the proteinwas still within the gel matrix to prevent further adsorptive lossesduring the initial lypholization step. Rehydration and in-gel digestionwas carried out in H₂ ¹⁸O containing a large excess of trypsin which wasalso reconstituted in H₂ ¹⁸O. During the digestion procedure, trypticpeptides liberated from the gel after the incorporation of the first ¹⁸Oatom can undergo the second carbonyl oxygen exchange process mediated bythe excess trypsin. This should promote the replacement of the secondcarbonyl oxygen since peptides liberated would have higher solubilitythan proteins thereby resulting in a higher level of doubly ¹⁸O labelledtryptic peptides (FIGS. 1 and 2; (54)). In order to prevent backexchange with normal water, trypsin was deactivated by boiling which hasbeen previously shown to be effective (51,54). In addition, the dried,deactivated mix was only resuspended and mixed immediately prior toinjection onto a nanoLC to minimize spontaneous exchange, although thisspontaneous exchange has been shown to be low (15,40).

Reverse Labelling

In the case of stable isotope labelling and quantification using MS,errors are potentially introduced during the labelling and ionizationprocess. These errors include the potential different affinity of thelabel and the possible suppression effect of the heavy or light labelledpeptides during the MALDI process (13,55). Traditional technicalreplicates which involve repeating the same labelling could result in anuncorrected bias towards a particular label or increased random error ofspecific peptides due to contaminating peaks. Our normal and reverselabelled technical replicates demonstrated a high degree of correlationwith scatter plot gradients of 0.97 (R²=0.92) and 0.93 (R²=0.82) forbiological replicates 1 and 2, respectively (FIG. 3) which is close tothe expected ratio of 1.0 for no labelling bias. These gradients alsoindicate that the method was reproducible with respect to proteinestimation, gel loading, gel excision and in-gel digestion. The lack ofbias suggests normalization routines like dye swap or LOWESS datanormalization routinely used in microarray experiments (35) might beunnecessary. However samples that are considerably more complex than thebacterial cell envelopes used in this study may still require reverselabelling validation as when one considers the influence of minorcontaminating peptides on the calculation of the ¹⁸O/¹⁶O ratios and theneed to verify peptides with extreme changes. The reverse-label designin addition to providing an estimate and means for correcting systematicerrors had the further benefit of allowing both the heavy and lightlabelled peptides to be readily identified since the MS/MS acquisitionmethod selected only the most intense peptide in each heavy/light pairto fragment. In this way the possibility of incorrect assignment isreduced. To our knowledge, this is the first report of reverse ¹⁶O/¹⁸Olabelling in a complex biological sample other than the recentquantitation of seventeen cytochrome P450 proteins (26,30).

Biofilm vs Planktonic Culture

We have demonstrated a strong positive correlation between thebiological replicates (r=0.701, p<0.0001) indicating that there wasreproducibility in biofilm formation and development. This was also seenby the finding that 70 out of 81 quantifiable proteins were observed toexhibit similar ratios in both biological replicates (Table 2, ranked 1or 2). More than three quarters of the P. gingivalis proteins identifiedin this study were identified by >2 unique peptides, further increasingthe confidence of identification and quantification of this labellingprocedure. Of the 81 proteins consistently identified from bothbiological replicates, 47 significantly changed in abundance from theplanktonic to biofilm state. The change in abundance of a percentage ofthe detected proteome, especially in the cell envelope, is consistentwith other studies on biofilm forming bacteria such as Pseudomonasaeruginosa, where over 50% of the detected proteome was shown to exhibitsignificant changes in abundance between planktonic and mature biofilmgrowth phases. (12). We further observed a wide range of responses inthe cell envelope proteome of P. gingivalis to growth as a biofilm. Anumber of proteins previously demonstrated to be altered in abundance inresponse to biofilm culture were also found to have changed in abundancein our study. Remarkably some proteins were observed to have changed inabundance by up to fivefold (Table 2) suggesting some major shifts inthe proteome in response to biofilm culture.

C-Terminal Domain Family

P. gingivalis has recently been shown to possess a novel family of up to34 cell surface-located outer membrane proteins that have no significantsequence similarities apart from a conserved C-Terminal Domain (CTD) ofapproximately 80 residues (31,56). The P. gingivalis CTD family ofproteins includes the gingipains (RgpA [PG2024], RgpB [PG0506], Kgp[PG1844]); Lys- and Arg-specific proteinases and adhesins, that aresecreted and processed to form non-covalent complexes on the cellsurface and are considered to be the major virulence factors of thisbacterium (57-61). Gingipains have been linked directly to diseasepathogenesis due to their ability to degrade host structural and defenseproteins and the inability of mutants lacking functional Kgp or RgpB tocause alveolar bone loss in murine periodontal models (62). Althoughthese CTD family proteins have a variety of functions the known andputative functions of the CTD family proteins are strongly focusedtowards adhesive and proteolytic activities and also include the CPG70carboxypeptidase (63), PrtT thiol proteinase, HagA haemagglutinin, S.gordonii binding protein (PG0350, (64)) a putative haemagglutinin,putative thiol reductase, putative fibronectin binding protein, putativeLys-specific proteinase (PG0553) and a putative von Willebrand factordomain protein amongst others. The majority of these proteins are likelyto play important roles in the virulence of the bacterium as they areinvolved in extracellular proteolytic activity, aggregation, haem/ironcapture and storage, biofilm formation and maintenance, virulence andresistance to oxidative stress. The CTD has been proposed to play rolesin the secretion of the proteins across the outer membrane and theirattachment to the surface of the cell, probably via glycosylation(56,65,66). In this work we were able to quantify nine CTD familyproteins consistently regulated in both replicates (Table 2) and allexcept PG2216 and PG1844 (Kgp) had increased in abundance during thebiofilm state. The significant increase in the abundance of many of thisgroup of proteins therefore suggests they play important functionalroles during the biofilm state.

The major cell surface proteases of P. gingivalis RgpA, Kgp are known tobe actively involved in peptide and haem acquisition, especially fromhaemoglobin and the release of haem at the cell surface (67,68). Duringthe biofilm state, there was an average 2.7 fold increase in theabundance of RgpA. HagA which contains the adhesin domains that are alsofound in RgpA and Kgp that are responsible for haemagglutination andhemoglobin binding of P. gingivalis (69) was also higher in abundance inthe biofilm state.

Kgp in contrast was observed to be significantly lower in abundance inbiofilm cells of P. gingivalis. This could be due to a decrease in Kgpabundance or may be due to the release of Kgp from the P. gingivaliscell surface during biofilm culture. Kgp is essential for P. gingivalisto hydrolyze haemoglobin at surface-exposed Lys residues which leads tothe release and uptake of peptides and haem (67,70). The adhesiondomains of Kgp are involved in haemoglobin binding and Genco et al (70)have proposed that Kgp acts as a haemophore, that like siderophores, isreleased from the cell surface to scavenge haem from the environment.Kgp with bound haem is then proposed to bind to HmuR, an outer membraneTonB-linked receptor, reported to be required for both haemoglobin andhaem utilization and deliver haem to the cell (71). Interestingly, HmuYa protein that is encoded in an operon with HmuR, was also more abundantin biofilm cultured cells.

The hmu locus contains 6 genes (hmuYRSTUV) and has been suggested tobelong to the multigenic cluster encoding proteins involved in thehaem-acquisition pathways similar to the Iht and Htr systems (72). HmuYwas shown to be required for both haemoglobin and haem utilization andis regulated by iron availability (72,73). Although HmuR was notidentified in our study, the operonic nature of hmuR and hmuY and otherevidence suggests that their expression is similarly regulated and theyact in concert for haem utilization (71,74). The decrease in abundanceof Kgp and the increase in abundance of HmuY is therefore consistentwith its proposed role as a hemophore and haem limitation in biofilmgrowth (see below).

CPG70 (PG0232) a CTD family protease that has been demonstrated to beinvolved in gingipain processing was also consistently higher inabundance in biofilm culture possibly indicating a role in theremodeling of cell surface proteins during biofilm growth (63,75). A CTDfamily putative thioredoxin (PG0616) was also significantly higher inabundance in the biofilm state. PG0616 has been characterized as HBP35,a haem binding protein having coaggregation properties (76). Ofparticular note was the increased abundance of the immunoreactive 46 kDaantigen, PG99 by an average factor of 5.0 in biofilm cells (Table 2).This was the highest observed increase in protein abundance in thisstudy, and since PG99 is both immunogenic and a CTD family member andtherefore most probably located on the cell surface, this proteinrepresents a good potential target for biofilm disruptive agents.

Transport Proteins

Two putative TonB dependent receptor family proteins (PG1414 and PG2008)and a putative haem receptor protein (PG1626) also show significantincrease in abundance. The exact functions of these proteins areunknown, however a COG search on the NCBI COG database resulted in hitsto the P functional class of outer membrane receptor proteins involvingmostly Fe transport (77). Interestingly we also observed an increasedabundance of the intracellular iron storage protein ferritin (PG1286).The consistent increases in abundance of these iron/haem transportingand storage proteins could be an indication of haem/iron limitation,especially within the deeper layers of the biofilm since ferritin isimportant for P. gingivalis to survive under iron-depleted conditions(78).

It is likely that both haemoglobin and haem would not diffuse far intothe biofilm due to the high proteolytic activity, high haemoglobin andhaem binding and storage capacities of P. gingivalis. It is alsopossible that ferrous iron transport via FeoB1 plays a more importantrole in the iron metabolism of this species in the deeper layers of thebiofilm which may also explain the increase in ferritin as there wouldbe little chance of cell surface storage of iron as haem (45,79). P.gingivalis grown under conditions of haem limitation exhibits anincrease in intracellular iron, indicating that PPIX is the growthlimiting factor and that ferrous iron is accumulated via the FeoB1transporter (45).

IhtB (PG0669) and a putative TonB dependent receptor (PG0707) bothshowed a decrease in abundance in the biofilm state (Table 2). IhtB is ahaem binding lipoprotein that also has been proposed to function as aperipheral outer membrane chelatase that removes iron from haem prior touptake by P. gingivalis (80). A similar decrease in abundance of twoproteins potentially involved in haem/Fe uptake during the biofilm statethat coincided with an increase in abundance of many others indicates ashift in either the types of receptors being used for uptake or morelikely a change in the substrate being used. Taken together from theabove observations, it appears that P. gingivalis growing in a biofilmis likely to be haem starved. The higher abundance of some transport andbinding proteins therefore suggests them being more crucial during thebiofilm state and thus possible antimicrobial drug targets.

There is a higher abundance of the glycolytic enzyme glyceraldehyde3-phosphate dehydrogenase (GAPDH) during the biofilm state compared tothe planktonic which is consistent with previous results obtained forListeria monocytogenes and Pseudomonas aeruginosa (12,106). AlthoughGAPDH is classified as a tetrameric NAD-binding enzyme involved inglycolysis and gluconeogenesis, there have been numerous reports of thisprotein being multifunctional and when expressed at the cell surface ofGram-positive bacteria, it appeared to be involved in binding ofplasmin, plasminogen and transferrin (107,108). Interestinglycoaggregation between Streptococcus oralis and P. gingivalis 33277 hasbeen shown to be mediated by the interaction of P. gingivalis fimbriaeand S. oralis GAPDH (109). The exact role, if any, of GAPDH in substratebinding in P. gingivalis however remains to be answered.

Biofilm Formation

There was a significantly higher abundance of the universal stressprotein (UspA) in the planktonic cells as compared to the biofilm cells.The production of Usp in various bacteria was found to be stimulated bya large variety of conditions, such as entry into stationary phase,starvation of certain nutrients, oxidants and other stimulants(110,111). The increased abundance in planktonic phase cells isconsistent with the fact that P. gingivalis has evolved to grow as partof a biofilm and that planktonic phases are likely to be more stressful.Expression of UspA in P. gingivalis is thought to be related to biofilmformation as inactivation of uspA resulted in the attenuation of earlybiofilm formation by planktonic cells (112). In this study the biofilmhas been established and reached maturation, it therefore appears tohave lesser need for UspA as compared to free floating planktonic cells.

A homologue of the internalin family protein InIJ (PG0350) was observedto be higher in abundance during the biofilm state. PG0350 has beenshown to be important for biofilm formation in P. gingivalis 33277 asgene inactivation resulted in reduced biofilm formation (39). Higherlevels of PG0350 in the biofilm could suggest that this protein might berequired not just for initial biofilm formation but acts an adhesin thatbinds P. gingivalis to each other or extracellular substrates within thebiofilm.

Proteins with Unknown Functions

The largest group of proteins identified in this study was 41 proteinswith unknown functions including four proteins that were identified forthe first time in this study (Table 2). Of the 41 proteins identified,37 were predicted to be from the cell envelope and within this group 17proteins show significant changes between the biofilm and planktoniccells. The majority of these proteins have homology to GenBank proteinswith defined names but not well-defined functions. Of particularinterest are several proteins that were consistently found tosubstantially increase in abundance in the biofilm state, namely PG0181,PG0613, PG1304, PG2167 and PG2168.

The above results represent a large scale validation of the ¹⁶O/¹⁸Oproteolytic labelling method as applied to a complex mixture, and arethe first to use this approach for the comparison of bacterial biofilmand planktonic growth states. A substantial number of proteins with avariety of functions were found to consistently increase or decrease inabundance in the biofilm cells, indicating how the cells adapt tobiofilm conditions and also providing potential targets for biofilmcontrol strategies.

TABLE 1 Quantification of predetermined BSA ratios using ¹⁶O/¹⁸Oproteolytic labelling Mean ratio Expected ratio 1:1 a)Triplicate analysis (±SD) CCTESLVNR 0.83 0.84 0.88 0.85 ± 0.03 DLGEEHFK0.95 1.06 0.85 0.95 ± 0.10 EACFKVEGPK 1.09 1.12 1.09 1.10 ± 0.02ECCDKPLLEK 1.01 0.96 0.87 0.94 ± 0.07 EYEATLEECCAK 1.05 1.01 1.05 1.04 ±0.02 LVTDLTKVHK 0.86 0.91 1.02 0.93 ± 0.08 RHPEYAVSVLLR 1.07 0.96 0.940.99 ± 0.07 YICDNQDTISSK 1.00 1.15 1.03 1.06 ± 0.08 Average 0.98 ± 0.101.00 ± 0.10 0.97 ± 0.09 0.98 ± 0.08 Average of all peptides ID** 0.98 ±0.12 CV of all peptides 13.1% Expected ratio 2:1 b)Expected ratio 1:2 b) 18O/16O 18O/16O QTALVELLK 1.92 0.44 (2.27) 2.10LVNELTEFAK 2.45 0.46 (2.17) 2.31 RHPEYAVSVLLR 1.82 0.42 (2.36) 2.09LGEYGFQNALIVR 2.21 0.43 (2.31) 2.26 MPCTEDYLSLILNR 2.59 0.40 (2.50) 2.55KVPQVSTPTLVEVSR 2.35 0.39 (2.57) 2.46 LFTFHADICTLPDTEK 1.72 0.44 (2.27)2.00 RPCFSALTPDETYVPK 1.82 0.52 (1.92) 1.87 Average 2.11 ± 0.33 2.30 ±0.20 2.24 ± 0.24 Average of all peptides ID*** 2.22 ± 0.26CV of all peptides ID 11.75% Expected ratio 1:5 Expected ratio 10:1(18O/16O) (18O/16O) AEFVEVTK 0.232 (4.32) AEFVEVTK 12.38 CCTESLVNR0.184 (5.42) QTALVELLK 10.40 SHCIAEVEK 0.176 (5.67) LVNELTEFAK 14.17ECCDKPLLEK 0.169 (5.91) FKDLGEEHFK  9.41 HPEYAVSVLLR 0.218 (4.58)HPEYAVSVLLR 11.76 YICDNQDTISSK 0.187 (5.36) YICDNQDTISSK 10.16LKECCDKPLLEK 0.252 (3.97) RHPEYAVSVLLR 10.14 SLHTLFGDELCK 0.183 (5.45)SLHTLFGDELCK  7.58 RHPEYAVSVLLR 0.201 (4.97) EYEATLEECCAK 14.07VPQVSTPTLVEVSR 0.206 (4.86) ETYGDMADCCEK 12.67 ECCHGDLLECADDR0.298 (3.35) LGEYGFQNALIVR  9.36 LFTFHADICTLPDTEK 0.210 (4.76)VPQVSTPTLVEVSR  8.34 KVPQVSTPTLVEVSR  8.86 LFTFHADICTLPDTEK 11.08RPCFSALTPDETYVPK 10.26 Average 0.210 ± 0.04 (4.90 ± 0.75) 10.74 ± 2.04CV of all peptides ID 15.26% 18.95% a) For expected ratio of 1:1, onlypeptides that were identified in all three separate experiments areincluded in this table b) For expected ratio of 2:1 and 1:2, onlypeptides that were identified in both experiments are included in thistable **n = 55 ***n = 24

TABLE 2 List of the 81 proteins identified from both biologicalreplicates of the P. gingivalis cell envelope fraction. An abundanceratio of >1 indicates a higher abundance of the protein in the biofilmwith respect to the planktonic state. If the ratio differs from one withmore than 3-fold SD (0.26) from the predetermined BSA ratios (>1.78 or<0.56), the proteins were considered to have significantly changed.Based on their mean ratios, proteins highlighted in grey representsignificant changes.

a Locations as determined by the CELLO program; EX: Extracellular, OM:Outer membrane, IM: Inner membrane, PP: Periplasm, CY: Cytoplasm; UN:unknown b Maximum Mascot peptide ion score / identity threshold cNormalized ratio; B = Biofilm, P = Planktonic, Normalization process asdescribed in experimental procedures d SE = Standard error of the mean eRanking and grouping as described in FIG. 4B f Proteins identified onlyin this study ! SE measurements not carried out due tounresolved/overlapping of one of the 3 peptides * Due to presence ofidentical peptides between these proteins, ratios derived were frompeptides that were unique to these proteins only. Values in parenthesisare total number of peptides matched. ** Due to unresolved/overlappingpeaks

TABLE 3 Proteomic and transcriptomic analyses of genes products involvedin glutamate/aspartate catabolism in P. gingivalis during growth inmeme-limitation compared to heme-excess. Shading indicates proteins thatare predicted to be encoded in operons.

¹Highest scoring peptide score/threshold score (P = 0.05) ²Total numberof independent peptide identification events for each protein ³Number ofunique ICAT-labelled peptides identified for each protein ⁴Averageratios of all quantified peptides for each protein in fold change(Heme-limitation/excess) ⁵NS no statistically significant changedetected ⁶Only identified in the microarray analysis C*DenotesICAT-modified cysteine

TABLE 4 The 24 P. gingivalis polypeptides selected as targets forinhibition of biofilm formation.

* These accession numbers provide a sequence for the P. gingivalisproteins referred to in the specification. Sequences corresponding tothe accession numbers are incorporated by reference.

TABLE 5 Proteomic and transcriptomic analyses of P. gingivalis grown inheme-limitation compared to heme-excess. Shading indicates proteins thatare predicted to be encoded in operons.

¹Highest scoring peptide score/threshold score (P = 0.05) ²Total numberof independent peptide identification events for each protein ³Number ofunique ICAT-labelled peptides identified for each protein ⁴Averageratios of all quantified peptides for each protein in fold change(Heme-limitation/excess) ⁵NS no statistically significant changedetected ⁶Only identified in microarray analysis C*Denotes ICAT-modifiedcysteine

REFERENCES

-   1. Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D.    R., and Lappin-Scott, H. M. (1995) Annu. Rev. Microbiol. 49, 711-745-   2. Cvitkovitch, D. G., Li, Y. H., and Ellen, R. P. (2003) J. Clin.    Invest. 112(11), 1626-1632-   3. Cochrane, D. M., Brown, M. R., Anwar, H., Weller, P. H., Lam, K.,    and Costerton, J. W. (1988) J. Med. Microbiol. 27(4), 255-261-   4. van Steenbergen, T. J., Kastelein, P., Touw, J. J., and de    Graaff, J. (1982) J. Periodontal Res. 17(1), 41-49-   5. Neiders, M. E., Chen, P. B., Suido, H., Reynolds, H. S.,    Zambon, J. J., Shlossman, M., and Genco, R. J. (1989) J. Periodontal    Res. 24(3), 192-198-   6. Griffen, A. L., Becker, M. R., Lyons, S. R., Moeschberger, M. L.,    and Leys, E. J. (1998) J. Clin. Microbiol. 36(11), 3239-3242-   7. Cutler, C. W., Arnold, R. R., and Schenkein, H. A. (1993) J.    Immunol. 151(12), 7016-7029-   8. Chen, T., Hosogi, Y., Nishikawa, K., Abbey, K., Fleischmann, R.    D., Walling, J., and Duncan, M. J. (2004) J. Bacteriol. 186(16),    5473-5479-   9. Davey, M. E. (2006) Periodontol. 2000 42, 27-35-   10. Orme, R., Douglas, C. W., Rimmer, S., and Webb, M. (2006)    Proteomics 6(15), 4269-4277-   11. Rathsam, C., Eaton, R. E., Simpson, C. L., Browne, G. V.,    Valova, V. A., Harty, D. W., and Jacques, N. A. (2005) J Proteome    Res 4(6), 2161-2173-   12. Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W., and    Davies, D. G. (2002) J. Bacteriol. 184(4), 1140-1154-   13. Ong, S. E., and Mann, M. (2005) Nat. Chem. Biol. 1(5), 252-262-   14. Bender, M. L., and Kemp, K. C. (1957) J. Am. Chem. Soc 79, 116-   15. Schnolzer, M., Jedrzejewski, P., and Lehmann, W. D. (1996)    Electrophoresis 17(5), 945-953-   16. Yao, X., Freas, A., Ramirez, J., Demirev, P. A., and    Fenselau, C. (2001) Anal Chem 73(13), 2836-2842-   17. Blonder, J., Hale, M. L., Chan, K. C., Yu, L. R., Lucas, D. A.,    Conrads, T. P., Zhou, M., Popoff, M. R., Issaq, H. J., Stiles, B.    G., and Veenstra, T. D. (2005) J. Proteome Res 4(2), 523-531-   18. Qian, W. J., Monroe, M. E., Liu, T., Jacobs, J. M., Anderson, G.    A., Shen, Y., Moore, R. J., Anderson, D. J., Zhang, R., Calvano, S.    E., Lowry, S. F., Xiao, W., Moldawer, L. L., Davis, R. W.,    Tompkins, R. G., Camp, D. G., 2nd, and Smith, R. D. (2005) Mol. Cell    Proteomics 4(5), 700-709-   19. Zang, L., Palmer Toy, D., Hancock, W. S., Sgroi, D. C., and    Karger, B. L. (2004) J. Proteome Res. 3(3), 604-612-   20. Kuster, B., and Mann, M. (1999) Anal. Chem. 71(7), 1431-1440-   21. Takao, T., Hori, H., Okamoto, K., Harada, A., Kamachi, M., and    Shimonishi, Y. (1991) Rapid Commun. Mass Spectrom. 5(7), 312-315-   22. Shevchenko, A., Chemushevich, I., Ens, W., Standing, K. G.,    Thomson, B., Wilm, M., and Mann, M. (1997) Rapid Commun. Mass    Spectrom. 11(9), 1015-1024-   23. Gevaert, K., Staes, A., Van Damme, J., De Groot, S., Hugelier,    K., Demol, H., Martens, L., Goethals, M., and    Vandekerckhove, J. (2005) Proteomics 5(14), 3589-3599-   24. Chen, X., Cushman, S. W., Pannell, L. K., and Hess, S. (2005) J.    Proteome Res. 4(2), 570-577-   25. Stockwin, L. H., Blonder, J., Bumke, M. A., Lucas, D. A.,    Chan, K. C., Conrads, T. P., Issaq, H. J., Veenstra, T. D.,    Newton, D. L., and Rybak, S. M. (2006) J. Proteome Res. 5(11),    2996-3007-   26. Lane, C. S., Wang, Y., Betts, R., Griffiths, W. J., and    Patterson, L. H. (2007) Mol. Cell Proteomics-   27. Korbel, S., Schumann, M., Bittorf, T., and Krause, E. (2005)    Rapid Comm. Mass Spectrom. 19(16), 2259-2271-   28. Bantscheff, M., Dumpelfeld, B., and Kuster, B. (2004) Rapid    Commun. Mass. Spectrom. 18(8), 869-876-   29. Jia, J. Y., Lamer, S., Schumann, M., Schmidt, M. R., Krause, E.,    and Haucke, V. (2006) Mol. Cell Proteomics 5(11), 2060-2071-   30. Miyagi, M., and Rao, K. C. (2007) Mass Spectrom. Rev 26(1),    121-136-   31. Veith, P. D., Talbo, G. H., Slakeski, N., Dashper, S. G., Moore,    C., Paolini, R. A., and Reynolds, E. C. (2002) Biochem. J. 363(Pt    1), 105-115-   32. Qian, W. J., Liu, T., Monroe, M. E., Strittmatter, E. F.,    Jacobs, J. M., Kangas, L. J., Petritis, K., Camp, D. G., 2nd, and    Smith, R. D. (2005) J. Proteome Res 4(1), 53-62-   33. Perkins, D. N., Pappin, D. J., Creasy, D. M., and    Cottrell, J. S. (1999) Electrophoresis 20(18), 3551-3567-   34. Xia, Q., Hendrickson, E. L., Zhang, Y., Wang, T., Taub, F.,    Moore, B. C., Porat, I., Whitman, W. B., Hackett, M., and    Leigh, J. A. (2006) Mol. Cell Proteomics 5(5), 868-881-   35. Quackenbush, J. (2001) Nat. Rev. Genet. 2(6), 418-427-   36. Yu, C. S., Chen, Y. C., Lu, C. H., and Hwang, J. K. (2006)    Proteins 64(3), 643-651-   37. Richardson, A. J., Calder, A. G., and Stewart, C. S. (1989)    Letters in Applied Microbiology 9, 5-8-   38. O'Toole, G. A., and Kolter, R. (1998) Mol Microbiol 28(3),    449-461-   39. Capestany, C. A., Kuboniwa, M., Jung, I. Y., Park, Y.,    Tribble, G. D., and Lamont, R. J. (2006) Infect. Immun. 74(5),    3002-3005-   40. Lopez-Ferrer, D., Ramos-Fernandez, A., Martinez-Bartolome, S.,    Garcia-Ruiz, P., and Vazquez, J. (2006) Proteomics 6 Suppl 1, S4-S11-   41. Staes, A., Demol, H., Van Damme, J., Martens, L.,    Vandekerckhove, J., and Gevaert, K. (2004) J Proteome Res 3(4),    786-791-   42. Patwardhan, A. J., Strittmatter, E. F., Camp, D. G., 2nd,    Smith, R. D., and Pallavicini, M. G. (2006) Proteomics 6(9),    2903-2915-   43. Smalley, J. W., Birss, A. J., McKee, A. S., and    Marsh, P. D. (1993) J Gen Microbiol 139(9), 2145-2150-   44. McKee, A. S., McDermid, A. S., Baskerville, A., Dowsett, A. B.,    Ellwood, D. C., and Marsh, P. D. (1986) Infect. Immun. 52(2),    349-355-   45. Dashper, S. G., Butler, C. A., Lissel, J. P., Paolini, R. A.,    Hoffmann, B., Veith, P. D., O'Brien-Simpson, N. M., Snelgrove, S.    L., Tsiros, J. T., and Reynolds, E. C. (2005) J. Biol. Chem.    280(30), 28095-28102-   46. Li, J., Steen, H., and Gygi, S. P. (2003) Mol. Cell Proteomics    2(11), 1198-1204-   47. Dashper, S. G., Brownfield, L., Slakeski, N., Zilm, P. S.,    Rogers, A. H., and Reynolds, E. C. (2001) J. Bacteriol. 183(14),    4142-4148-   48. Hoskisson, P. A., and Hobbs, G. (2005) Microbiology 151(Pt 10),    3153-3159-   49. Piper, M. D., Daran-Lapujade, P., Bro, C., Regenberg, B.,    Knudsen, S., Nielsen, J., and Pronk, J. T. (2002) J. Biol. Chem.    277(40), 37001-37008-   50. Siroy, A., Cosette, P., Seyer, D., Lemaitre-Guillier, C.,    Vallenet, D., Van Dorsselaer, A., Boyer-Mariotte, S., Jouenne, T.,    and De, E. (2006) J. Proteome Res. 5(12), 3385-3398-   51. Hood, B. L., Lucas, D. A., Kim, G., Chan, K. C., Blonder, J.,    Issaq, H. J., Veenstra, T. D., Conrads, T. P., Pollet, I., and    Karsan, A. (2005) J Am Soc Mass Spectrom 16(8), 1221-1230-   52. Yao, X., Afonso, C., and Fenselau, C. (2003) J Proteome Res    2(2), 147-152-   53. Eckel-Passow, J. E., Oberg, A. L., Therneau, T. M., Mason, C.    J., Mahoney, D. W., Johnson, K. L., Olson, J. E., and Bergen, H. R.,    3rd. (2006) Bioinformatics 22(22), 2739-2745-   54. Storms, H. F., van der Heijden, R., Tjaden, U. R., and van der    Greef, J. (2006) Rapid Commun. Mass Spectrom. 20(23), 3491-3497-   55. Zenobi, R., and Knochenmuss, R. (1998) Mass Spectrom. Rev.    17(5), 337-366-   56. Seers, C. A., Slakeski, N., Veith, P. D., Nikolof, T., Chen, Y.    Y., Dashper, S. G., and Reynolds, E. C. (2006) J. Bacteriol.    188(17), 6376-6386-   57. Curtis, M. A., Kuramitsu, H. K., Lantz, M., Macrina, F. L.,    Nakayama, K., Potempa, J., Reynolds, E. C., and    Aduse-Opoku, J. (1999) J. Periodontal Res. 34(8), 464-472-   58. O'Brien-Simpson, N. M., Paolini, R. A., Hoffmann, B., Slakeski,    N., Dashper, S. G., and Reynolds, E. C. (2001) Infect. Immun.    69(12), 7527-7534-   59. O'Brien-Simpson, N. M., Veith, P. D., Dashper, S. G., and    Reynolds, E. C. (2003) Curr. Protein Pept. Sci. 4(6), 409-426-   60. Abe, N., Kadowaki, T., Okamoto, K., Nakayama, K., Ohishi, M.,    and Yamamoto, K. (1998) J. Biochem. (Tokyo) 123(2), 305-312-   61. Potempa, J., Pike, R., and Travis, J. (1995) Infect. Immun.    63(4), 1176-1182-   62. Pathirana, R. D., O'Brien-Simpson, N. M., Brammar, G. C.,    Slakeski, N., and Reynolds, E. C. (2007) Infect. Immun. 75(3),    1436-1442-   63. Chen, Y. Y., Cross, K. J., Paolini, R. A., Fielding, J. E.,    Slakeski, N., and Reynolds, E. C. (2002) J. Biol. Chem. 277(26),    23433-23440-   64. Zhang, Y., Wang, T., Chen, W., Yilmaz, O., Park, Y., Jung, I.    Y., Hackett, M., and Lamont, R. J. (2005) Proteomics 5(1), 198-211-   65. Sato, K., Sakai, E., Veith, P. D., Shoji, M., Kikuchi, Y.,    Yukitake, H., Ohara, N., Naito, M., Okamoto, K., Reynolds, E. C.,    and Nakayama, K. (2005) J. Biol. Chem. 280(10), 8668-8677-   66. Nguyen, K. A., Travis, J., and Potempa, J. (2007) J. Bacteriol.    189(3), 833-843-   67. Dashper, S. G., Cross, K. J., Slakeski, N., Lissel, P., Aulakh,    P., Moore, C., and Reynolds, E. C. (2004) Oral Microbiol. Immunol.    19(1), 50-56-   68. Lewis, J. P., Dawson, J. A., Hannis, J. C., Muddiman, D., and    Macrina, F. L. (1999) J. Bacteriol. 181(16), 4905-4913-   69. Shi, Y., Ratnayake, D. B., Okamoto, K., Abe, N., Yamamoto, K.,    and Nakayama, K. (1999) J. Biol. Chem. 274(25), 17955-17960-   70. Sroka, A., Sztukowska, M., Potempa, J., Travis, J., and    Genco, C. A. (2001) J. Bacteriol. 183(19), 5609-5616-   71. Simpson, W., Olczak, T., and Genco, C. A. (2000) J Bacteriol    182(20), 5737-5748-   72. Lewis, J. P., Plata, K., Yu, F., Rosato, A., and    Anaya, C. (2006) Microbiology 152(Pt 11), 3367-3382-   73. Olczak, T., Siudeja, K., and Olczak, M. (2006) Protein Expr.    Purif. 49(2), 299-306-   74. Olczak, T., Simpson, W., Liu, X., and Genco, C. A. (2005) FEMS    Microbiol. Rev. 29(1), 119-144-   75. Veith, P. D., Chen, Y. Y., and Reynolds, E. C. (2004) Infect.    Immun. 72(6), 3655-3657-   76. Shibata, Y., Hiratsuka, K., Hayakawa, M., Shiroza, T.,    Takiguchi, H., Nagatsuka, Y., and Abiko, Y. (2003) Biochem. Biophys.    Res. Commun. 300(2), 351-356-   77. Tatusov, R. L., Fedorova, N. D., Jackson, J. D., Jacobs, A. R.,    Kiryutin, B., Koonin, E. V., Krylov, D. M., Mazumder, R.,    Mekhedov, S. L., Nikolskaya, A. N., Rao, B. S., Smirnov, S.,    Sverdlov, A. V., Vasudevan, S., Wolf, Y. I., Yin, J. J., and    Natale, D. A. (2003) BMC Bioinformatics 4, 41-   78. Ratnayake, D. B., Wai, S, N., Shi, Y., Amako, K., Nakayama, H.,    and Nakayama, K. (2000) Microbiology 146 1119-1127-   79. Smalley, J. W., Birss, A. J., McKee, A. S., and    Marsh, P. D. (1991) FEMS Microbiol. Lett. 69(1), 63-67-   80. Dashper, S. G., Hendtlass, A., Slakeski, N., Jackson, C.,    Cross, K. J., Brownfield, L., Hamilton, R., Barr, I., and    Reynolds, E. C. (2000) J Bacteriol 182(22), 6456-6462-   81. Takahashi, N., Sato, T., and Yamada, T. (2000) J. Bacteriol.    182(17), 4704-4710-   82. Takahashi, N., and Sato, T. (2001) J Dent Res 80(5), 1425-1429-   83. Litwin, C. M., and Calderwood, S. B. (1993) Clin Microbiol Rev    6(2), 137-149-   84. Gygi, S. P., Rochon, Y., Franza, B. R., and Aebersold, R. (1999)    Mol. Cell Biol. 19(3), 1720-1730-   85. Baughn, A. D., and Malamy, M. H. (2003) Microbiology 149(Pt 6),    1551-1558-   86. Macy, J., Probst, I., and Gottschalk, G. (1975) J Bacteriol    123(2), 436-442-   87. Baughn, A. D., and Malamy, M. H. (2002) Proc Natl Acad Sci USA    99(7), 4662-4667-   88. Mayrand, D., and McBride, B. C. (1980) Infect Immun 27(1), 44-50-   89. Smith, M. A., Mendz, G. L., Jorgensen, M. A., and    Hazell, S. L. (1999) Int J Biochem Cell Biol 31(9), 961-975-   90. Kroger, A., Geisler, V., Lemma, E., Theis, F., and    Lenger, R. (1992) Archives of Microbiology 158(5), 311-314-   91. Shah, H., and Williams, R. (1987) Current Microbiology 15,    241-246-   92. Klein, R. A., Linstead, D. J., and Wheeler, M. V. (1975)    Parasitology 71(1), 93-107-   93. Turrens, J. F. (1989) Biochem J 259(2), 363-368-   94. Mendz, G. L., Hazell, S. L., and Srinivasan, S. (1995) Arch    Biochem Biophys 321(1), 153-159-   95. Mendz, G. L., Meek, D. J., and Hazell, S. L. (1998) J Membr Biol    165(1), 65-76-   96. Mileni, M., MacMillan, F., Tziatzios, C., Zwicker, K., Haas, A.    H., Mantele, W., Simon, J., and Lancaster, C. R. (2006) Biochem J    395(1), 191-201-   97. Nealson, K., and D, S. (1994) Annual review of microbiology 48,    311-343-   98. Sellars, M. J., Hall, S. J., and Kelly, D. J. (2002) J Bacteriol    184(15), 4187-4196-   99. O'Toole, G. A., Gibbs, K. A., Hager, P. W., Phibbs, P. V., Jr.,    and Kolter, R. (2000) J Bacteriol 182(2), 425-431-   100. Whiteley, M., Bangera, M. G., Bumgarner, R. E., Parsek, M. R.,    Teitzel, G. M., Lory, S., and Greenberg, E. P. (2001) Nature    413(6858), 860-864-   101. Romeo, T., Gong, M., Liu, M. Y., and    Brun-Zinkernagel, A. M. (1993) J Bacteriol 175(15), 4744-4755-   102. Sabnis, N. A., Yang, H., and Romeo, T. (1995) J Biol Chem    270(49), 29096-29104-   103. Mercante, J., Suzuki, K., Cheng, X., Babitzke, P., and    Romeo, T. (2006) J Biol Chem 281(42), 31832-31842-   104. Altier, C., Suyemoto, M., and Lawhon, S. D. (2000) Infect Immun    68(12), 6790-6797-   105. Lawhon, S. D., Frye, J. G., Suyemoto, M., Porwollik, S.,    McClelland, M., and Altier, C. (2003) Mol Microbiol 48(6), 1633-1645-   106. Hefford, M. A., D'Aoust, S., Cyr, T. D., Austin, J. W.,    Sanders, G., Kheradpir, E., and Kalmokoff, M. L. (2005) Can. J.    Microbiol. 51(3), 197-208-   107. Pancholi, V., and Fischetti, V. A. (1992) J. Exp. Med. 176(2),    415-426-   108. Taylor, J. M., and Heinrichs, D. E. (2002) Mol. Microbiol.    43(6), 1603-1614-   109. Maeda, K., Nagata, H., Yamamoto, Y., Tanaka, M., Tanaka, J.,    Minamino, N., and Shizukuishi, S. (2004) Infect. Immun. 72(3),    1341-1348-   110. Gustaysson, N., Diez, A., and Nystrom, T. (2002) Mol.    Microbiol. 43(1), 107-117-   111. Kvint, K., Nachin, L., Diez, A., and Nystrom, T. (2003) Curr.    Opin. Microbiol. 6(2), 140-145-   112. Kuramitsu, H. K., Chen, W., and Ikegami, A. (2005) J.    Periodontol. 76(11 Suppl), 2047-2051

1. A composition for use in raising an immune response to P. gingivalisin a subject, the composition comprising an amount effective to raise animmune response of at least one antigenic or immunogenic portion of apolypeptide corresponding to accession numbers selected from the groupconsisting of AAQ65462, AAQ65742, AAQ66991, AAQ65561, AAQ66831,AAQ66797, AAQ66469, AAQ66587, AAQ66654, AAQ66977, AAQ65797, AAQ65867,AAQ65868, AAQ65416, AAQ65449, AAQ66051, AAQ66377, AAQ66444, AAQ66538,AAQ67117 and AAQ67118.
 2. A composition of claim 1 wherein the portionhas an amino acid sequence that is substantially identical to at least50 amino acids of one of the polypeptides.
 3. A composition of claim 2wherein the polypeptide corresponds to an accession number selected fromthe group consisting of AAQ65462, AAQ66991, AAQ65561 and AAQ66831.
 4. Acomposition of claim 2 wherein the polypeptide corresponds to accessionnumber AAQ65742.
 5. A composition for use in raising an immune responseto P. gingivalis in a subject, the composition comprising amounteffective to raise an immune response of at least one polypeptide havingan amino acid sequence substantially identical to at least 50 aminoacids of a polypeptide expressed by P. gingivalis and that is predictedby the CELLO program to be extracellular.
 6. A composition for use inraising an immune response to P. gingivalis in a subject, thecomposition comprising an amount effective to raise an immune responseof at least one polypeptide having an amino acid sequence selectedsubstantially identical to at least 50 amino acids of a polypeptide thatcauses an immune response in a mouse or a rabbit.
 7. A method ofpreventing, inhibiting or treating a subject for periodontal diseasecomprising administering to the subject an effective amount ofcomposition according to claim
 1. 8. A method of preventing or treatinga subject for P. gingivalis infection comprising administering to thesubject a composition according to claim
 1. 9. An antibody raisedagainst an antigenic region of a polypeptide having an amino acidsequence, the sequence being substantially identical to at least 50amino acids of one of the polypeptides corresponding to accessionnumbers AAQ65462, AAQ65742, AAQ66991, AAQ65561, AAQ66831, AAQ66797,AAQ66469, AAQ66587, AAQ66654, AAQ66977, AAQ65797, AAQ65867, AAQ65868,AAQ65416, AAQ65449, AAQ66051, AAQ66377, AAQ66444, AAQ66538, AAQ67117 andAAQ67118.
 10. An antibody of claim 9 wherein the polypeptide correspondsto an accession number selected from the group consisting of AAQ65462,AAQ66991, AAQ65561 and AAQ66831.
 11. An antibody of claim 9 wherein thepolypeptide corresponds to an accession number selected from the groupconsisting of AAQ65742.